System for reproducing and modulating stability and turnover of RNA molecules

ABSTRACT

An in vitro system is provided that recapitulates regulated mRNA stability and turnover of exogenous RNA substrates. The system comprises a cell extract optionally depleted of activity of proteins that bind polyadenylate, and a target RNA sequence. This system is used for the identification of agents capable of modulating RNA turnover, as well as agents capable of modulating RNA turnover in the presence of RNA stability modifying agents.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from Provisional ApplicationSerial No. 60/086,675, filed May 26, 1998.

GOVERNMENTAL SUPPORT

[0002] The research leading to the present invention was supported, atleast in part, by grant No. GM56434 from the National Institutes forHealth. Accordingly, the Government may have certain rights in theinvention.

FIELD OF THE INVENTION

[0003] Broadly, the present invention involves a system and method formonitoring the stability of RNA and identifying agents capable ofmodulating RNA stability.

BACKGROUND OF THE INVENTION

[0004] The relative stability of a mRNA is an important regulator ofgene expression. The half-life of a mRNA plays a role in determiningboth the steady state level of expression as well as the rate ofinducibility of a gene product. In general, many short-lived proteinsare encoded by short-lived mRNAs. Several mRNAs that encode stableproteins, such as α-globin, have also been shown to have extraordinarilylong half-lives. Surveillance mechanisms are also used by the cell toidentify and shorten the half-lives of mRNAs that contain nonsense codonmutations. Clearly, changes in the half-life of a mRNA can have dramaticconsequences on cellular responses and function.

[0005] Little is known about mechanisms of mRNA turnover and stabilityin mammalian cells, but in vivo data are beginning to allow somegeneralizations about major pathways of mRNA turnover. The mRNA poly(A)tail can be progressively shortened throughout the lifetime of a mRNA inthe cytoplasm. Controlling the rate of this deadenylation processappears to be a target for many factors that regulate mRNA stability.Once the poly(A) tail is shortened to approximately 50-100 bases, thebody of the mRNA is degraded in a rapid fashion with no discernibleintermediates. The process of translation also influences mRNAstability. Little is known, however, concerning the enzymes andregulatory components involved in mammalian mRNA turnover.

[0006] Several cis-acting elements have been shown to play a role inmRNA stability. Terminal (5′) cap and 3′-poly(A) structures andassociated proteins are likely to protect the transcript fromexonucleases. Several destabilizing as well as stabilizing elementslocated in the body of the mRNA have also been identified. The bestcharacterized instability element is an A-U rich sequence (ARE) found inthe 3′ untranslated region of many short-lived mRNAs. These AREsprimarily consist of AUUUA (SEQ ID NO: 12) repeats or a relatednonameric sequence. AREs have been shown to increase the rate ofdeadenylation and mRNA turnover in a translation-independent fashion.For example, proteins with AU-rich elements include many growth factorand cytokine mRNAs, such as c-fos, c-jun, c-myc TNFα, GMCSF, IL1-15, andIFN-β. Other stability elements include C-rich stabilizing elements,such as are found in the mRNAs of globin, collagen, lipoxygenase, andtyrosine hydroxylase. Still other mRNAs have as yet uncharacterized orpoorly characterized sequence elements, for example, that have beenidentified by deletion analysis, e.g. VEGF mRNA.

[0007] Numerous proteins have been described that interact with somespecificity with an ARE, but their exact role in the process of mRNAturnover remains to be defined. For example, proteins which bind to theARE described above include HuR and other ELAv family proteins, such asHuR (also called HuA), He1-N1 (also called HuB), HuC and HuD; AUF1 (fourisoforms); tristetrapolin; AUH; TIA; TIAR; glyceraldehyde-3-phosphate;hnRNP C; hnRNP A1; AU-A; and AU-B. Many others have not been extensivelycharacterized.

[0008] Through the application of genetics, the mechanisms and factorsinvolved in the turnover of mRNA in Saccharomyces cerevisiae arebeginning to be identified. One major pathway of mRNA decay involvesdecapping followed by the action of a 5′-to-3′ exonuclease. Evidence hasalso been obtained for a role for 3′-to-5′ exonucleases in analternative pathway. Functionally significant interactions between thecap structure and the 3′ poly(A) tail of yeast mRNAs have also beendescribed. Several factors involved in the translation-dependent pathwayof nonsense-codon-mediated decay have also been identified. Whetherthese observations are generally applicable to mammalian cells, however,remains to be established.

[0009] Mechanistic questions in mammalian cells are usually bestapproached using biochemical systems due to the inherent difficultieswith mammalian cells as a genetic system. Thus, efforts have been madeto develop in vitro systems to study mRNA stability and turnover.However, the presently available in vitro systems suffer from numerouslimitations. For example, many suffer from poor data quality and ageneral lack of reproducibility that significantly limits theirapplication. Another key problem is that most of these systems do notfaithfully reproduce all aspects of mRNA stability. A significantdifficulty in the development of these systems is to differentiatebetween random, non-specific RNA degradation and true, regulated mRNAturnover. The significance of all previous in vitro systems to the truein vivo process of mRNA stability, therefore, is unclear. To date, no invitro mRNA stability system has been generally accepted in the field asvalid and useful. Other problems that have been uncovered in presentlyavailable systems are that they usually involve a complicated extractprotocol that is not generally reproducible by other laboratories in thefield. Also, presently available systems can only be used to assess thestability of endogenous mRNAs, severely limiting their utility. Finally,the data quality obtained using such systems is highly variable,precluding their use in sensitive screening assays.

[0010] Accordingly, there exists a need for an in vitro RNA stabilitysystem is efficient and highly reproducible, and further, one whichproduces minimal to undetectable amounts of RNA degradation

[0011] A further need exists for an in vitro RNA stability systemwherein deadenylation of an RNA transcript in the system should occurbefore general degradation of the mRNA body is observed. Also needed isan in vitro RNA stability system wherein degradation of the mRNA bodyoccurs in an apparently highly processive fashion without detectableintermediates, and further, the regulation of the rate of overalldeadenylation and degradation should be observed in a sequence-specificmanner. Such a system should be applicable to exogenous RNAs and allowease of experimental manipulation.

[0012] The citation of any reference herein should not be construed asan admission that such reference is available as “Prior Art” to theinstant application.

SUMMARY OF THE INVENTION

[0013] In accordance with the present invention, an in vitro system formodulating the stability and turnover of an RNA molecule is providedwhich models RNA processing in vivo. Thus, the present invention permitshigh throughput screening of compounds/macromolecules that modulate thestability of eukaryotic RNAs in order to identify and design drugs toaffect the expression of selected transcripts, as well as to aid in thecharacterization of endogenous proteins and other macromoleculesinvolved in mRNA stability. The in vitro system of the present inventionis useful as a diagnostic aid for determining the molecular defect inselective disease alleles; development of in vitro mRNA stabilitysystems for other eukaryotic organisms including parasites and fungiwhich should lead to novel drug discovery; and improving gene deliverysystems by using the system to identify factors and RNA sequences thataffect RNA stability.

[0014] Broadly, the present invention extends to an in vitro systemcapable of recapitulating regulated RNA turnover of an exogenously addedpreselected target RNA sequence, the system comprising a cell extractand a target RNA sequence. In a non-limiting example of the systemdescribed herein, the regulated RNA turnover is AU-rich elementregulated RNA turnover or C-rich element regulated RNA turnover.

[0015] The cell extract of the system of the present invention isisolated from lysed eukaryotic cells or tissues; the cell extract may beobtained for example from a cell line, such as HeLa cells or a T cellline, but the invention is not so limited. The cell extract may beprepared from cells comprising foreign nucleic acid, such as those thatare infected, stably transfected, or transiently transfected. The cellextract may be partially purified.

[0016] In one embodiment of the invention, the cell extract may bedepleted of activity of proteins that bind polyadenylate. The depletionof activity of proteins that bind polyadenylate from the cell extractmay be achieved by any of a number of methods, for example, the additionto the system of polyadenylate competitor RNA; the sequestration ofproteins that bind polyadenylate; the addition of a proteinase thatinactivates a protein that bind to polyadenylate; or addition of anagent that prevents the interaction between polyadenylate and anendogenous macromolecule that binds to polyadenylate, to name a few. Asfurther examples of the methods for sequestration of proteins that bindpolyadenylate, it may be achieved by such non-limiting procedures as thetreatment of the extract with an material that depletes macromoleculesthat bind polyadenylate, such as antibodies to proteins that bindpolyadenylate, polyadenylate, and the combination. The material may beattached to a matrix. Other methods to achieve the depletion of theactivity of proteins that bind polyadenylate may be used.

[0017] The target RNA sequence used in the system may be, by way ofnon-limiting examples, synthetic RNA, naturally occurring RNA, messengerRNA, chemically modified RNA, or RNA-DNA derivatives. The target RNAsequence may have a 5′ cap and a 3′ polyadenylate sequence. The targetRNA sequence may be unlabeled target RNA sequence, labeled target RNAsequence, or a the combination of both. The labeled RNA sequence may belabeled with a moiety such as, but not limited to a fluorescent moiety,a visible moiety, a radioactive moiety, a ligand, and a combination offluorescent and quenching moieties. Other moieties and means forlabeling RNA are embraced herein.

[0018] The system of the present invention may additionally includeexogenously added nucleotide triphosphate; ATP is preferred. It may alsoinclude a reaction enhancer to enhance the interaction between thevarious components present in the system, for example, polymers such asbut not limited to polyvinyl alcohol, polyvinylpyrrolidone and dextran;polyvinyl alcohol is preferred.

[0019] The present invention is also directed to a method foridentifying agents capable of modulating the stability of a target RNAsequence. The method is carried out by preparing the system describedabove which includes the cell extract depleted of activity of proteinsthat bind polyadenylate and the target RNA sequence; introducing intothe aforesaid system an agent to be tested; determining the extent ofturnover of the target RNA sequence by, for example, determining theextent of degradation of the labeled target RNA; and then identifying anagent which is able to modulate the extent of RNA turnover as capable ofmodulating the stability of the target RNA sequence.

[0020] The method described above may additionally include nucleotidetriphosphate, ATP being preferred. The agent to be tested may be, but isnot limited to, an RNA stability modifying molecule. The non-limitingselection of the types of target RNA sequence and the non-limiting typesof labels useful for the RNA as described hereinabove.

[0021] The method of the present invention is useful for identifyingagents which can either increases or decrease the stability of saidtarget RNA sequence. Such agents may be capable of modulating theactivity of an RNA binding molecule such as, but not limited to, C-richelement binding proteins and AU rich element binding proteins, examplesof the latter including HuR and other ELAv family proteins, such as HuR,He1-N1, HuC and HuD; AUF1; tristetrapolin; AUH; TIA; TIAR;glyceraldehyde-3-phosphate; hnRNP C; hnRNP A1; AU-A; and AU-B. This listis provided as illustrative of the types of molecules that may beevaluated in the present invention, but is by no means limiting.

[0022] In a further embodiment of the present invention, a method isprovided for identifying an agent that is capable of modulating thestability of a target RNA sequence in the presence of an exogenouslyadded RNA stability modifier or RNA binding macromolecule. Non-limitingexamples of such molecules are described above. The method is carriedout by preparing the system described above which includes the cellextract can be depleted of activity of proteins that bind polyadenylateand the target RNA sequence; introducing into the aforesaid system theexogenously added RNA stability modifier or binding macromolecule andthe agent to be tested; determining the extent of turnover of the targetRNA sequence by, for example, determining the extent of degradation ofthe labeled target RNA; and then identifying an agent able to modulatingthe extent of the RNA turnover as capable of modulating the stability ofthe target RNA sequence in the presence of the exogenously added RNAstability modifier.

[0023] The non-limiting selection of the components of this method areas described above. The aforementioned method is useful, for example,when the RNA stability modifier decreases the stability of said targetRNA sequence, and the agent to be identified increases the stability ofthe target RNA sequence that is decreased by the RNA stability modifier.In addition, the method is useful when the RNA stability modifierincreases the stability of the target RNA sequence, and the agent to beidentified decreases the stability of the target RNA sequence that isincreased by the RNA stability modifier. Non-limiting examples of RNAstability modifiers include C-rich element binding proteins, and AU richelement binding proteins, examples of AU rich element binding proteins,including HuR and other ELAv family proteins, such as HuR, He1-N1, HuCand HuD; AUF1; tristetrapolin; AUH; TIA; TIAR;glyceraldehyde-3-phosphate; hnRNP C; hnRNP A1; AU-A; and AU-B. This listis provided as illustrative of the types of molecules that may beevaluated in the present invention, but is by no means limiting.

[0024] The present invention is further directed to a method foridentifying an agent capable of modulating the deadenylation of a targetRNA sequence comprising preparing the system described above in theabsence of nucleotide triphosphate, such as ATP; introducing an agentinto the system; and monitoring the deadenylation of the target RNAsequence. Furthermore, the invention is also directed towards a methodfor identifying an agent capable of modulating the deadenylation anddegradation of a target RNA sequence comprising preparing the systemdescribed herein in the presence of ATP; introducing the agent into thesystem; and monitoring the deadenylation and degradation of the targetRNA sequence. These embodiments may also be carried out in the presenceof an RNA stability modifier or RNA binding macromolecule to determinethe ability of the agent to modulate the effect of the modulator orbinding molecule on RNA stability.

[0025] It is a further aspect of the present invention to provide amethod for identifying an agent capable of modulating cell growth orcell differentiation in a mammal comprising determining the ability ofsaid agent to modulate the stability of a target RNA sequence involvedin the modulation of cell growth or differentiation in accordance withthe methods described above. The agents capable of modulating cellgrowth or cell differentiation may intervene in such physiologicalprocesses as cellular transformation and immune dysregulation, but theinvention is not so limiting.

[0026] It is yet a further aspect of the present invention to provide amethod for identifying, characterizing and isolating an endogenousmolecule suspected of participating in the deadenylation or degradationof RNA or regulation thereof comprising preparing the system describedhereinabove; introducing a protein suspected of participating in theregulation of RNA turnover into said system; and monitoring thestability of the target RNA sequence. The endogenous molecule suspectedof participating in the deadenylation and/or degradation of RNA orregulation may be protein or RNA.

[0027] In another embodiment of the invention, a method is provided foridentifying an agent capable of modulating the degradation a target RNAsequence in the absence of deadenylation comprising providing a cellextract in the presence of a nucleotide triphosphate; introducing saidagent into said cell extract; and monitoring the degradation of saidtarget RNA sequence in said extract.

[0028] A further aspect of the present invention is directed to a kitfor monitoring the stability of a preselected target RNA sequence underconditions capable of recapitulating regulated RNA turnover. The kitcomprises a cell extract that optionally may be depleted of activity ofproteins that bind polyadenylate; other reagents; and directions foruse. The kit may further comprise nucleotide triphosphates, a reactionenhancer, or both.

[0029] Accordingly, it is an object of the invention to provide a systemfor modulating the stability and turnover of an RNA molecule in vitro,which permits a skilled artisan to study the turnover generally, ordeadenylation and degradation specifically, of an RNA transcript, andscreen drugs which can modulate the stability and turnover of an RNAtranscript. The turnover may be in the absence or presence ofexogenously added RNA stability modulators, or permit the study of therole of endogenous molecules in RNA turnover.

[0030] It is another embodiment of the invention to provide a kit that askilled artisan can readily use to modulate the stability and turnoverof an RNA molecule in vitro, and investigate the aforementioned agents.

[0031] These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIGS. 1A-D: The addition of poly(A) to cytoplasmic S100 extractsactivates specific deadenylase and degradation activities. Panel A.Poly(A) competitor RNA activates nucleolytic activities in the extract.A capped, radiolabeled 54 base RNA containing a 60 base poly(A) tail(Gem-A60) was incubated at 30° C. with S100 extract in the absence(lanes marked S100) or presence (Lanes marked S100+Poly(A)) of 500 ng ofcold poly(A) RNA as described in Materials and Methods of Example I forthe times indicated. RNA products were analyzed on a 5% acrylamide gelcontaining 7M urea. The position of a deadenylated, 54 base transcript(Gem-A0) is indicated on the right. Panel B. The shortening of inputtranscripts is due to a 3′-to-5′ exonuclease. Gem-A60 RNA, labeledexclusively at the 5′ cap, was incubated in the in vitro mRNA stabilitysystem for the times indicated. Reaction products were analyzed on a 5%acrylamide gel containing 7M urea. The position of a deadenylated, 54base transcript (Gem-A0) is indicated on the right. Panel C. Analternative approach also demonstrates that the shortening of inputtranscripts is due to a 3′-to-5′ exonuclease. ARE-A60 RNA, radiolabeledat A residues, was incubated in the in vitro stability system for thetimes indicated. Reaction products were hybridized to a DNA oligo andcleaved into 5′ and 3′ fragments using RNase H. Fragments were analyzedon a 5% acrylamide gel containing 7M urea. Panel D. The 3′-to-5′exonuclease activity is a specific deadenylase. Gem-A60 RNA or a variantthat contains 18 extra nucleotides after the poly(A) tract (Gem-A60-15)were incubated in the in vitro stability system for the times indicated.RNA products were analyzed on a 5% acrylamide gel containing 7M urea.The position of a deadenylated, 54 base transcript (Gem-A0) is indicatedon the left. 31±11.0% of the input Gem-A60 RNA was deadenylated/degradedin 30 min.

[0033] FIGS. 2A-E: The rate of transcript degradation in the in vitrosystem is regulated by AU-rich instability elements in asequence-specific fashion. Panel A. AU-rich elements dramaticallyincrease the rate of turnover in the in vitro system. Gem-A60 RNA or apolyadenylated transcript that contains the 34 base AU-rich element fromthe TNF-α mRNA, were incubated in the in vitro stability system for thetimes indicated. RNA products were analyzed on a 5% acrylamide gelcontaining 7M urea. The positions of deadenylated transcripts (Gem-A0and ARE-A0) are indicated. The ARE-A60 RNA was deadenylated/degraded6.6±0.4 fold faster than Gem-A60 RNA. Panel B. The AU-rich element fromc-fos mRNA also functions as an instability element in vitro. Gem-A60RNA or a transcript that contains the 72 base AU-rich element from thec-fos mRNA (Fos-A60) were incubated in the in vitro stability system forthe times indicated. RNA products were analyzed on a 5% acrylamide gelcontaining 7M urea. The positions of deadenylated transcripts (Gem-A0and Fos-A0) are indicated. The Fos-A60 RNA was deadenylated/degraded3.5±0.3 fold faster than Gem-A60 RNA. Panel C. The ability of AU-richelements to mediate transcript instability in the in vitro system issequence-specific. ARE-A60 RNA or a variant that contains a mutation atevery fourth position (mt ARE-A60; see Materials and Methods) wereincubated in the in vitro stability system for the times indicated. RNAproducts were analyzed on a 5% acrylamide gel containing 7M urea. Thepositions of deadenylated transcripts (ARE-A0 and mt ARE-A0) areindicated. Mutations in the ARE reduced the rate ofdeadenylation/degradation by 3.7±1.4 fold compared to the wild typeARE-A60 transcript. Panel D. The TNF-α AU-rich element mediatesinstability in a heterologous context. A polyadenylated 250 base RNAderived from the SV late transcription unit (SV-A60), or a variant thatcontains the 34 base AU-rich element from the TNF-α mRNA (SVARE-A60),were incubated in the in vitro stability system for the times indicated.RNA products were analyzed on a 5% acrylamide gel containing 7M urea.The positions of deadenylated transcripts (SV-A0 and SVARE-A0) areindicated. SVARE-A60 RNA was deadenylated/degraded 3.5±0.7 fold fasterthan SV-A60 RNA. Panel E. The AU-rich element derived from the GM-CSFmRNA functions in vitro on nearly a full length RNA substrate. A nearlyfull length version of the GM-CSF mRNA that contained an AU-rich element(GM−CSF(+ARE), or a version in which the AU-rich element was deleted(GM−CSF(−ARE), were incubated in the in vitro stability system for thetimes indicated. RNA products were analyzed on a 5% acrylamide gelcontaining 7M urea. GM−CSF(+ARE) was deadenylated/degraded 2.8±0.2 foldfaster than the GM−CSF(−ARE) transcript.

[0034] FIGS. 3A-B: Deadenylation occurs in the absence of ATP and isregulated by AU-rich elements in vitro. Panel A. Degradation, but notdeadenylation, requires ATP. SV-ARE-A60 RNA was incubated in the invitro system in the presence ((+) ATP lanes) or absence ((−) ATP lanes)for the times indicated. RNA products were analyzed on a 5% acrylamidegel containing 7M urea. The positions of the deadenylated SVARE-A)transcript is indicated. Panel B. AU-rich elements regulate the rate ofdeadenylation on RNA substrates which carry a physiologic length poly(A)tail. SV RNA or SV-ARE RNA (a variant that contains an AU-rich element)were polyadenylated with yeast poly(A) polymerase and species thatcontained tails of approximately 150-200 bases were gel purified. TheseRNAs (SV(A150-200) and SVARE(A150-200) were incubated in the in vitrostability system for the times indicated. RNA products were analyzed ona 5% acrylamide gel containing 7M urea. The positions of deadenylatedtranscripts (SV-A0 and SVARE-A0) are indicated. SVARE(A 150-200) RNA wasdeadenylated 2.2±0.3% fold faster than the SV(A150-200) transcript.

[0035] FIGS. 4A-B: The HuR protein of the ELAV family specifically bindsto the TNF-α AU-rich element in the in vitro system. Panel A. Twoproteins specifically interact with the TNF-α AU-rich element. Gem-A60and ARE-A60 RNAs were radiolabeled at U residues and incubated in the invitro stability system for 5 min. in the presence of EDTA (to blockdegradation and allow for accurate comparisons). Reaction mixtures wereirradiated with UV light, cleaved with RNase A, and protein-RNAcomplexes were analyzed on a 10% acrylamide gel containing SDS. Theapproximate sizes of the cross linked proteins indicated on the rightwere deduced from molecular weight markers. Panel B. The 30 kDa proteinis HuR. Radiolabeled ARE-A60 RNA was incubated in the in vitro RNAstability system and cross-linked to associated proteins as describedabove. Cross linked proteins were immunoprecipitated using the indicatedantisera prior to analysis on a 10% acrylamide gel containing SDS. Thelane marked Input denotes total cross linked proteins prior toimmunoprecipitation analysis.

[0036] FIGS. 5A-C: While AU-rich element binding factors are importantto promote RNA deadenylation and degradation, the binding of the HuRprotein to AU-rich elements is not associated with AU-richelement-mediated transcript instability. Panel A. Competition analysissuggests that AU-rich element binding factors are required fordeadenylation and degradation of transcripts. SVARE-A60 RNA wasincubated in the in vitro stability system for 30 min. in the presenceof the indicated amounts of a synthetic RNA competitor that containedthe TNF-α AU-rich element (ARE comp.) or a non-specific sequence. RNAproducts were analyzed on a 5% acrylamide gel containing 7M urea. Theposition of deadenylated SVARE-A0 RNA is indicated. Panel B. Reactionmixtures were prepared as described in panel A with the addition of EDTAto inhibit RNA turnover. Protein-RNA interactions were analyzed by UVcross linking analysis and analyzed on a 10% acrylamide gel containingSDS. The positions of AU rich element-specific cross linked species isindicated on the left. Panel C. Reactions were prepared exactly asdescribed for Panel B, except samples were immunoprecipitated usinga-HuR specific antisera prior to gel electrophoresis.

[0037] FIGS. 6A-D: ELAV proteins specifically stabilize deadenylatedintermediates in the in vitro system. Panel A. SVARE-A60 RNA wasincubated in the in vitro system in the presence (lanes(+) He1-N1)) orthe absence (lanes (−) He1-N1) of 1 ug of recombinant He1-N1 protein.RNA products were analyzed on a 5% acrylamide gel containing 7M urea.The position of deadenylated SVARE-A0 transcript is indicated. Panel B.SVARE-A60 RNA was incubated in the in vitro system in the presence of 1ug of recombinant He1-N1 (lanes (+) He1-N1), GST only (lanes (+) GST),or an unrelated RNA binding protein hnRNP H′ (lanes (+) hnRNP H′). RNAproducts were analyzed on a 5% acrylamide gel containing 7M urea. Theposition of deadenylated SVARE-A0 transcript is indicated. Panel C.ARE-A60 RNA, or an unrelated transcript that lacked an AU-rich element(CX-A60), were incubated in the in vitro stability system for 30min. inthe presence (+lanes) or absence (−lanes) of ˜1 ug of He1-N2 protein.RNA products were analyzed on a 5% acrylamide gel containing 7M urea.The positions of deadenylated transcripts are indicated. Panel D. Avariant of SV-A60 RNA that contained the TNF-α ARE in the 5′ portion ofthe transcript (SV5′AGE-A60) was incubated in the in vitro system for 50min in the absence (−lane) or presence (+lane) of 1 μg of He1-N2protein. RNA products were analyzed on a 5% acrylamide gel containing 7M urea. The positions of imput and deadenylated transcripts areindicated.

DETAILED DESCRIPTION OF THE INVENTION

[0038] Numerous terms and phrases are used throughout the instantSpecification. The meanings of these terms and phrases are set forthbelow.

[0039] In particular, as used herein “half-life” of an RNA moleculerefers to the measurement of the decline in the amount of an RNAmolecule to serve as a template for the synthesis of its proteinproduct.

[0040] As used herein “turnover” refers to the degradation of an RNAmolecule. Turnover comprises deadenylation and degradation.

[0041] As used herein a “cap” or “5′ cap” or “terminal cap”, and be usedinterchangeable, and refer to a 7-methyl guanosine (7 mG) cap chemicallyconjugated to the most 5′ nucleotide of the RNA molecule.

[0042] As used herein, the phrase “polyadenylic acid (poly(A)) tail”refers to a string of contiguous adenylic acids (polyadenylate) addedpost transcriptionally to the 3′ end of an RNA molecule, such as mRNA.

[0043] As used herein, the term “stability” refers to the maintenance ofan RNA molecule so that it can function, and thus retard the degradationprocess of an RNA molecule.

[0044] As used herein, the phrase “a polyadenylic acid competitornucleic acid oligomer” refers to an oligomer comprising contiguousadenylic acids” which can be added to a system of the invention andsequester proteins that bind poly(A). Thus, the degradation of aparticular RNA molecule having a poly(A) tail can be modulated.

[0045] Also, as used herein, the phrase “restriction endonuclease”refers to an enzyme that recognizes specific nucleotide sequences in anucleic acid molecule, and produces a double-stranded break within ornear the site. Some restriction enzymes, such as EcoRI or HindIIIproduce “complementary tails” on each of fragments produced. These tailsare said to be “sticky” because under hybridization conditions they canreanneal with each other. Thus, if two separate nucleic acid moleculesshare the same restriction site, then both will contain complementarysingle-stranded tails when treated with the same restrictionendonuclease, and can be spliced together forming a recombinant nucleicacid molecule.

[0046] Naturally, as used herein, the phrase “restriction endonucleasesite” refers to a specific nucleotide sequence that is recognized by aspecific restriction endonuclease.

[0047] Furthermore, numerous conventional molecular biology,microbiology, and recombinant DNA techniques within the skill of the artcan be readily utilized to practice the instant invention. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, SecondEdition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: A PracticalApproach, Volumes I and II (D. N. Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames& S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames& S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed.(1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

[0048] Therefore, if appearing herein, the following terms shall havethe definitions set out below.

[0049] A “vector” is a replicon, such as plasmid, phage or cosmid, towhich another DNA segment may be attached so as to bring about thereplication of the attached segment. A “replicon” is any genetic element(e.g., plasmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo, i.e., capable of replication under its owncontrol.

[0050] A “cassette” refers to a segment of a nucleic acid molelcule,such as DNA or RNA, that can be inserted into a vector at specificrestriction sites. The segment of the nucleic acid molelcule may encodea polypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

[0051] A cell has been “transfected” by exogenous or heterologous DNAwhen such DNA has been introduced inside the cell. A cell has been“transformed” by exogenous or heterologous DNA when the transfected DNAeffects a phenotypic change. Preferably, the transforming DNA should beintegrated (covalently linked) into chromosomal DNA making up the genomeof the cell.

[0052] A “nucleic acid molecule” refers to the phosphate ester polymericform of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranologs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

[0053] A DNA “coding sequence” is a double-stranded DNA sequence whichis transcribed and translated into a polypeptide in a cell in vitro orin vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxyl) terminus. A coding sequence can include, but is notlimited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomicDNA sequences from eukaryotic (e.g., mammalian) DNA, and even syntheticDNA sequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

[0054] A “promoter sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

[0055] The present invention is based upon Applicant's discovery of aheretofore unknown system for activating regulated turnover of RNAmolecules in vitro that surprisingly and unexpectedly permits a skilledartisan to study and to modulate the stability and thus the turnover ofa RNA molecule in vitro. Thus, the new and useful system of theinvention permits accurate and faithful reproduction of both general andregulated aspects deadenylation and degradation of an RNA molecule, alsoreferred to herein as recapitulating regulated RNA turnover,particularly a eukaryotic mRNA transcript. In particular, the new anduseful system of the invention permits minimal amounts, preferablyundetectable, of mRNA turnover, and further, deadenylation of an RNAmolecule occurs in the system prior to degradation of the RNA molecule,which mimics the turnover process of RNA found in vivo.

[0056] The key to the development of the system and methods utilizingthe system are based on the discovery that polyadenylate competitor RNAis capable of sequestering proteins that bind polyadenylate andconsequently activating the deadenylase enzyme, inducing RNA turnover.As it was heretofore considered that such proteins that bindpolyadenylate may contribute to RNA deadenylation, the present findingthat such proteins are, in contrast, stabilizers of RNA, led to therealization that the such proteins are interacting with and inactivatingdestabilizing mediators in vivo. Thus, the present invention is directedto an in vitro system capable of recapitulating regulated RNA turnoverof an exogenously added preselected target RNA sequence comprising acell extract depleted of activity of proteins that bind polyadenylate,and a preselected target RNA sequence. In one particular embodiment, theregulated RNA turnover is that modulated by AU-rich element (ARE)regulated RNA turnover. Examples of mRNAs with AU-rich elements includethose of, by way of non-limiting example, c-fos; c-jun; c-myc TNF-α,GMCSF, IL1-15, and IFN-β. As noted above, AU-rich elements are sites forbinding of numerous proteins, including the ELAV family of ARE-bindingproteins, such as HuR, He1-N1, HuC and HuD; others include AUF1;tristetrapolin; AUH; TIA; TIAR; glyceraldehyde-3-phosphate; hnRNP C;hnRNP A1; AU-A; and AU-B. In another embodiment, the regulated RNAturnover is that modulated by C-rich element (CRE) regulated RNAturnover, such elements as found in the mRNA of globin mRNAs, collagen,lipoxygenase, and tyrosine hydroxylase. Another mRNA with an as yetuncharacterized sequence element is that of VEGF. The invention,however, is not so limiting as to the particular elements or bindingproteins to these elements involved in the regulation of RNA turnover.

[0057] The cell extract of the present invention is prepared from lysedeukaryotic cells or tissues. Various methods known to the skilledartisan may be used to prepare the cell extract. Various sources ofcells may be used, including fresh cells and tissues, and cells lines.Such cells may comprise foreign nucleic acid, such as in cells that areinfected; or are transiently or stably transfected with a mammalianexpression vector, the latter as described in more detail below. Forcertain purposes, for example to investigate the role of infection, andin particular intracellular infection, on RNA turnover, infected cellsmay be utilized as the source of the cell extract herein. Cells infectedwith viruses or other intracellular microorganisms such as Listeriamonocytogenes, HTLV, herpes simplex virus, and HIV, may be employed forthese particular circumstances. Furthermore, prior to preparation of thecell extract, cells may be exposed to certain chemical or otherextracellular stimuli, for example, hormones, growth factors, and kinaseand phosphatase inhibitors, which may alter RNA turnover, for whichsubsequent studies as described herein may be used to identify theinduction of certain proteins involved in modulating RNA turnover, orfor the identification of agents which may counteract adverse RNAturnover modulation induced by such stimuli. As will be noted in moredetail below, the methods herein may be used to identify agents whichmay protect cells by interfering with adverse RNA turnover induced byvarious sources. The cell extract is preferably free of nuclei andnuclear contents and comprises cytoplasm, but this is not essentialunless particular components, such as enzymes or other factors, fromnuclei, interfere with the operation of the system. In a typicalpreparation, which may be modified without departing from the scope ofthe invention, cells are grown, harvested, lysed, centrifuged for100,000×g for 1 hour, and dialyzed. Glycerol may be added to protect theextract if stored frozen.

[0058] As described above, a cell used to prepare the cell extract maycomprise foreign DNA. An isolated nucleic acid molecule to placed in asystem of the invention can initially be inserted into a cloning vectorto produce numerous copies of the molecule. A large number ofvector-host systems known in the art may be used. Possible vectorsinclude, but are not limited to, plasmids or modified viruses, but thevector system must be compatible with the host cell used. Examples ofvectors include, but are not limited to, E. coli, bacteriophages such aslambda derivatives, or plasmids such as pBR322 derivatives or pUCplasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc. Theinsertion into a cloning vector can, for example, be accomplished byligating the nucleic acid molecule into a cloning vector which hascomplementary cohesive termini. However, if the complementaryrestriction sites used to fragment the nucleic acid molecule are notpresent in the cloning vector, the ends of the molecule may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the termini of thenucleic acid molecule; these ligated linkers may comprise specificchemically synthesized oligonucleotides encoding restrictionendonuclease recognition sequences. Recombinant molecules can beintroduced into host cells via transformation, transfection, infection,electroporation, etc., so that many copies of the nucleic acid moleculeare generated. Preferably, the cloned nucleic acid molecule is containedon a shuttle vector plasmid, which provides for expansion in a cloningcell, e.g., E. coli, and facile purification for subsequent insertioninto an appropriate expression cell line, if such is desired. Forexample, a shuttle vector, which is a vector that can replicate in morethan one type of organism, can be prepared for replication in both E.coli and Saccharomyces cerevisiae by linking sequences from an E. coliplasmid with sequences from the yeast 2μ plasmid.

[0059] Naturally, any of the methods previously described for theinsertion of an isolated nucleic acid molecule into a cloning vector maybe used to construct expression vectors containing a nucleic acidmolecule consisting of appropriate transcriptional/translational controlsignals and the protein coding sequences. These methods may include invitro recombinant DNA and synthetic techniques and in vivo recombination(genetic recombination).

[0060] Mammalian expression vectors contemplated for use in theinvention include vectors with inducible promoters, such as thedihydrofolate reductase (DHFR) promoter, e.g., any expression vectorwith a DHFR expression vector, or a DHFR/methotrexate co-amplificationvector, such as pED (PstI, SalI, SbaI, SmaI, and EcoRI cloning site,with the vector expressing both the cloned gene and DHFR; see Kaufman,Current Protocols in Molecular Biology, 16.12 (1991). Alternatively, aglutamine synthetase/methionine sulfoximine co-amplification vector,such as pEE14 (HindIII, XbaI, SmaI, SbaI, EcoRI, and BclI cloning site,in which the vector expresses glutamine synthase and the cloned gene;Celltech). In another embodiment, a vector that directs episomalexpression under control of Epstein Barr Virus (EBV) can be used, suchas pREP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnIcloning site, constitutive RSV-LTR promoter, hygromycin selectablemarker; Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII,NheI, PvuII, and KpnI cloning site, constitutive hCMV immediate earlygene, hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI,NheI, HindIII, NotI, XhoI, SfiI, BamH1 cloning site, induciblemetallothionein IIa gene promoter, hygromycin selectable marker:Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloningsite, RSV-LTR promoter, histidinol selectable marker; Invitrogen), pREP9(KpnI, NheI, HindIII, NotI, XhoI, SfiI, and BamHI cloning site, RSV-LTRpromoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTRpromoter, hygromycin selectable marker, N-terminal peptide purifiablevia ProBond resin and cleaved by enterokinase; Invitrogen). Selectablemammalian expression vectors for use in the invention include pRc/CMV(HindIII, BstXI, NotI, SbaI, and ApaI cloning site, G418 selection;Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site,G418 selection; Invitrogen), and others. Vaccinia virus mammalianexpression vectors (see, Kaufman, 1991, supra) for use according to theinvention include but are not limited to pSC11 (SmaI cloning site, TK-and β-gal selection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI,ApaI, NheI, SacII, KpnI, and HindIII cloning site; TK- and β-galselection), and pTKgptF1S (EcoRI, PstI, SalI, AccI, HindII, SbaI, BamHI,and Hpa cloning site, TK or XPRT selection).

[0061] Once a particular nucleic acid molecule, such as RNA, is insertedinto a vector, several methods known in the art may be used to propagateit. Once a suitable host system and growth conditions are established,recombinant expression vectors can be propagated and prepared inquantity. As previously explained, the expression vectors which can beused include, but are not limited to, the following vectors or theirderivatives: human or animal viruses such as vaccinia virus oradenovirus; insect viruses such as baculovirus; yeast vectors;bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNAvectors, to name but a few. In addition, a host cell strain may bechosen which modulates the expression of the inserted sequences, ormodifies and processes the gene product in the specific fashion desired.Vectors are introduced into the desired host cells by methods known inthe art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0062] Cells useful for the preparation described herein includeimmortalized or partially immortalized cells which can be grown in largeamounts under defined conditions, such as HeLa cells and various T-cellcell lines. Other sources include tissues, blood cells, or myeloidcells. Other sources are well within the realm of the present invention.

[0063] The cell extract of the system described herein is depleted ofactivity of proteins that bind polyadenylate. This may be achieved byany one or a combination of methods such as the following. While notbeing bound by theory, each of these methods either removes the proteinsthat bind polyadenylate, or inactivate the binding activity. Theseprocedures may be applied to the cell extract as it is used in themethods described herein, or the cell extract may be treated beforehand.For example, a polyadenylate competitor RNA may be added to the cellextract to provide an irrelevant RNA sequence to which the bindingproteins may bind, thus clearing the target RNA sequence of such bindingproteins. In another embodiment, sequestration of proteins that bindpolyadenylate may be performed. Sequestration may be achieved by addingto the cell extract or exposing the cell extract to a material thatbinds the aforementioned proteins, such as antibodies to proteins thatbind polyadenylate, or polyadenylate sequences themselves ormacromolecules comprising polyadenylate sequences which serve as bindingtargets for such proteins. Alternatively or in addition, these proteinbinding materials may be bound to a matrix, such as agarose beads, andthe cell extract passed through a column of such beads to remove theproteins which bind polyadenylate. The preparation of such beadscovalently modified to comprise antibodies or RNA sequences, whetherpolyadenylate or sequences comprising polyadenylate, are known to theskilled artisan. Another means for reducing or eliminating such activityfrom the cell extract is by exposure to one or more proteinase known toinactivate a protein that bind to polyadenylate. These proteinases maybe added to the extract, or bound to a matrix and exposed to theextract, after which inactivation the beads may be removed. A furthermeans encompasses addition to the extract of an agent that prevents theinteraction between polyadenylate and an endogenous macromolecule thatbinds to polyadenylate. These and other methods embraced by the presentinvention achieve the desired goal of depleting macromolecules that bindpolyadenylate from the cell extract, thus allowing the cell extract incombination with the target RNA sequence to undergo in vivo-like RNAturnover. One or a combination of the aforesaid methods may be employedto reduce the level of such protein to an acceptable limit, dependentupon the source of the cells or tissues from which the extract is made,the particular target RNA sequence, and other factors. As will be notedbelow, certain macromolecules that bind to polyadenylate may be includedin particular screening assays or other methods employing the system andmethods described herein when that particular protein or othermacromolecule is subject to investigation as described herein.

[0064] In a further embodiment of the invention, the cell extract may bepartially purified or otherwise manipulated. For example, the cellextract may be partially purified to remove certain components beforebeing placed in the system of the invention, before or after beingoptionally depleted of macromolecules that bind polyadenylate. Forexample, certain non-specific factors and/or activities unrelated to ofinterfering with the methods of the present invention may be removedfrom the cell extract. The skilled artisan will recognize for theparticular target RNA being investigated hereunder the need for partialpurification of the extract and the need for depletion of factors thatbind polyadenylate. Furthermore, other components may be added to ensurethat the system of the invention recapitulates regulated RNA turnover.

[0065] The target RNA sequence in the system of the present inventionmay be an one of a number of RNA or modified RNA molecules. For example,synthetic RNA may be prepared by solid phase synthesis, or reproduced byin vitro transcription using phage polymerase as is known to the skilledartisan. Naturally occurring RNA may be isolated from cells, tissues,and other biological sources. The RNA may be a messenger RNA (mRNA), apreferred species herein, or RNA-DNA derivatives. Messenger RNAtypically comprises a 5′ cap and a 3′ polyadenylate sequence. Chemicallymodified RNA, such as RNA modified by phosphothioate moiety(ies), isembraced herein.

[0066] The particular RNA, including mRNA, used in the system andmethods of the present invention may be selected depending on theparticular species of mRNA to be studied. Investigations of mRNAturnover, endogenous modulators of its turnover and exogenously addedmolecules, particularly small molecules which affect mRNA turnover, haveimportant therapeutic implications in the prophylaxis and treatment of avariety of conditions and diseases. Certain mRNAs are short-lived, suchas those of cytokines; others are long-lived, such as globin message.The regulation of mRNA lifetimes for particular proteins and particularcell types may be subject to various adverse effects, from infection toexternal stimuli, which alter the turnover and hence cellularphysiology. In various conditions, altered expression of cellularproteins and cellular phenotypes may be consequences of altered mRNAturnover. Pharmacological intervention of such altered mRNA turnover, torestore an altered turnover, or the induction of an altered turnover toachieve a benefit to the organism, are achievable based upon the systemsand methods described herein. For example, a particular mRNA, such asthat of the proinflammatory cytokine TNFα, is selected as a target foridentification of small molecule modulators that may decrease theturnover, and this prolong the lifetime, and expression, of this proteinby inflammatory cells. Such modulators may provide substantial benefitin the treatment of certain immunological diseases wherein an increasedsecretion of TNFα is beneficial. Conversely, massive overproduction ofTNFα in sepsis, or its adverse effects in rheumatoid arthritis andinflammatory bowel disease may be ameliorated by use of an agent whichfurther increases the turnover ands thus decreases the expression ofTNFα by inflammatory cells.

[0067] The application of the invention herein to other mRNA species isembraced by the teachings herein. In particular, the methods of thepresent invention facilitate high throughput screening for theidentification of modulators of RNA turnover, to be applied to thetreatment or prophylaxis of disease.

[0068] One aspect of the system and method of the present invention ismonitoring the turnover of the target RNA sequence. This may be achievedby any one or a combination of various methods known to the skilledartisan, one of which is the provision of labeled RNA. The target RNAsequence of the present may be unlabeled, labeled, or a combination. Forexample, after setting up conditions under which the deadenylationand/or degradation of the unlabeled target RNA sequence occurs, itslevel may be assessed by any of a number of methods utilizing a labeledprobe, such as by hybridization, or by way of UV absorbance, gelelectrophoresis followed by specific or nonspecific staining, or usingan amplification system, such as phage polymerase, and then quantitationby a suitable amplification-based technique such as the molecular beaconmethod. Alternatively, and perhaps more simply, the target mRNA sequencemay be labeled, and the extent of intact sequence or degraded RNAfragments readily quantitated. Labels such as a fluorescent moiety, avisible moiety, a radioactive moiety, a ligand, and a combination offluorescent and quenching moieties. These non-limiting examples areprovided for purposes of illustration only.

[0069] Furthermore, optionally, an RNA molecule or a portion thereof,such as its poly(A) tail, may be detectably labeled using routineprotocols readily known to a skilled artisan. Suitable labels includeenzymes, fluorophores (e.g., fluorescein isothiocyanate (FITC),phycoerythrin (PE), Texas red (TR), rhodamine, free or chelatedlanthanide series salts, especially Eu³⁺, to name a few fluorophores),chromophores, radioisotopes, chelating agents, dyes, colloidal gold,latex particles, ligands (e.g., biotin), and chemiluminescent agents.When a control marker is employed, the same or different labels may beused for the receptor and control marker.

[0070] In the instance where a radioactive label, such as the isotopes³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and¹⁸⁶Re are used, known currently available counting procedures may beutilized. Particular ribonucleotides bay be prepared using theappropriate isotopes, and the labeled RNA prepared by solid phasesynthesis. Alternatively, moieties comprising the isotopes may becovalently bound to the RNA. In the instance where the label is anenzyme, detection may be accomplished by any of the presently utilizedcolorimetric, spectrophotometric, fluorospectrophotometric, amperometricor gasometric techniques known in the art. In a further example, biotinmoieties may be incorporated into the RNA by any number of means.Subsequently, the biotinylated RNA or degradation fragments may bequantitated by an avidin reagent;

[0071] Direct labels are one example of labels which can be usedaccording to the present invention. A direct label has been defined asan entity, which in its natural state, is readily visible, either to thenaked eye, or with the aid of an optical filter and/or appliedstimulation, e.g. U.V. light to promote fluorescence. Among examples ofcolored labels, which can be used according to the present invention,include metallic sol particles, for example, gold sol particles such asthose described by Leuvering (U.S. Pat. No. 4,313,734); dye soleparticles such as described by Gribnau et al. (U.S. Pat. No. 4,373,932)and May et al. (WO 88/08534); dyed latex such as described by May,supra, Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated inliposomes as described by Campbell et al. (U.S. Pat. No. 4,703,017).Other direct labels include a radionucleotide, a fluorescent moiety or aluminescent moiety. In addition to these direct labelling devices,indirect labels comprising enzymes can also be used according to thepresent invention. Various types of enzyme linked iminunoassays are wellknown in the art, for example, alkaline phosphatase and horseradishperoxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactatedehydrogenase, urease, these and others have been discussed in detail byEva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods inEnzymology, 70. 419-439, 1980 and in U.S. Pat. No. 4,857,453.

[0072] Suitable enzymes include, but are not limited to, alkalinephosphatase and horseradish peroxidase. Other labels for use in theinvention include magnetic beads or magnetic resonance imaging labels.

[0073] As noted herein, turnover of RNA occurs in two steps:deadenylation, which is not dependent upon the presence of nucleotidetriphosphates, and degradation, which is so dependent. The level ofnucleoside triphosphates, including ribonucleotide and/ordeoxyribonucleotide triphosphates, ATP, UTP, CTP, TTP, and/or GTP, inthe cell extract may or may not be sufficient to permit the degradationaspect of RNA turnover to occur. In one embodiment of the presentinvention, the system described herein additionally comprisesexogenously added nucleotide triphosphate, preferably ATP.

[0074] It was noted during the development of the present invention thatthe inclusion of a reaction enhancer resulted in a slight stimulation inthe efficiency of RNA degradation. This is likely to be due to itsability to promote macromolecular complex formation in vitro. Therefore,the invention herein optionally includes the use of a reaction enhancersuch as a polymer, to stimulate interaction among the components of thesystem. Non-limiting examples include polyvinyl alcohol,polyvinylpyrrolidone and dextran; polyvinyl alcohol is preferred.

[0075] The above-described system which recapitulates in vitro the RNAturnover of preselected RNA sequences has several utilities, inparticular, the identification of the role of endogenous factors andexogenous modulators in RNA turnover. The present invention is broadlydirected to a method for identifying an agent capable of modulating thestability of a target RNA sequence comprising

[0076] (A) preparing the system as described hereinabove;

[0077] (B) introducing said agent into said system;

[0078] (C) determining the extent of turnover of said target RNAsequence; and

[0079] (D) identifying an agent able to modulate the extent of saidturnover as capable of modulating the stability of said target RNAsequence.

[0080] The above method may additionally comprise added nucleotidetriphosphate, preferably ATP, for the purposes described above.

[0081] Agents whose activity in modulating RNA turnover may de detectedin the aforementioned method include but is not limited to an RNAstability modifying molecule.

[0082] As described above, the target RNA sequence may be selected asdescribed above, depending on the particular RNA to be studied. Thetarget RNA may be unlabeled target RNA sequence, labeled target RNAsequence, or the combination thereof. Labels include but are not limitedto a fluorescent moiety, a visible moiety, a radioactive moiety, aligand, or a combination of fluorescent and quenching moieties.

[0083] The monitoring the extent of turnover of said target RNA sequencecomprises determining the extent of degradation of said labeled targetRNA, by the methods described above.

[0084] In particular, the present method may be directed to identifyingagents capable of modulating the stability of a target RNA sequencewhich increases the stability of the target RNA sequence, oralternatively, decreasing the stability of the RNA sequence.

[0085] In a particular embodiment, the agent is capable of modulatingthe activity of a AU rich element binding protein or a C-rich element,but it is not so limited. Examples of AU rich element binding proteinsand C-rich element binding proteins are as described herein.

[0086] In a further embodiment of the present invention, a method isprovided for identifying an agent capable of modulating the stability ofa target RNA sequence in the presence of an exogenously added RNAstability modifier comprising

[0087] (a) preparing the system as described hereinabove;

[0088] (b) introducing said RNA stability modifier into said system;

[0089] (c) introducing said agent into said system;

[0090] (d) determining the extent of turnover of said target RNAsequence; and

[0091] (e) identifying an agent able to modulate the extent of saidturnover as capable of modulating the stability of said target RNAsequence in the presence of said exogenously added RNA stabilitymodifier.

[0092] This aspect of the invention is directed to identifying agents,in particular small molecules, capable of affecting the activity of aRNA turnover modulator. As described above, such small molecules may bescreened to determine their effect on the RNA stabilizing ordestabilizing ability of an endogenous mediator, which is added to thetest system. Alternatively, it may be used to identify compounds whichagonize or antagonize exogenous agents. The components of the system,including nucleotide triphosphate, the target RNA, labels, are asdescribed above. In one aspect of this embodiment, the RNA stabilitymodifier increases the stability of said target RNA sequence, and in afurther embodiment, the agent decreases the stability of said target RNAsequence increased by said RNA stability modifier. In anotherembodiment, the RNA stability modifier decreases the stability of saidtarget RNA sequence, and in a further embodiment, the agent increasesthe stability of said target RNA sequence decreased by said RNAstability modifier.

[0093] Candidate series of RNA stability modifiers include the AU richelement binding proteins, but the invention is not limited to suchfactors. Examples of known proteins having such elements in the mRNA,and binding proteins to the elements, are described above, however, theinvention is not limited to these examples.

[0094] Furthermore, in another embodiment, the macromolecules that bindRNA that are removed from the cell extract in accordance with theaforementioned procedures may be added back to the system herein toinvestigate their role in RNA turnover as well as the effect of agents,in particular small molecules, on RNA turnover modulated by thesemacromolecules that bind RNA. This embodiment may be applied to any ofthe methods described herein. In yet another embodiment, the target RNAmay be loaded with a macromolecule that binds RNA prior to addition tothe system herein, for the same purposes stated above.

[0095] As noted above, the cell extract used in any of the methodsdescribed herein may be partially purified.

[0096] A method is also provided for identifying an agent capable ofmodulating the deadenylation of a target RNA sequence comprising

[0097] (A) preparing the system of the present invention in the absenceof a nucleotide triphosphate;

[0098] (B) introducing said agent into said system; and

[0099] (C) monitoring the deadenylation of said target RNA sequence insaid system.

[0100] A further method is provided for identifying an agent capable ofmodulating the deadenylation and degradation of a target RNA sequencecomprising

[0101] (A) preparing the system of the present invention in the presenceof ATP;

[0102] (B) introducing said agent into said system; and

[0103] (C) monitoring the deadenylation and degradation of said targetRNA sequence in said system.

[0104] Method are also provided herein for identifying an agent capableof modulating cell growth or cell differentiation in a mammal comprisingdetermining the ability of said agent to modulate the stability of atarget RNA sequence involved in the modulation of cell growth ordifferentiation, utilizing the aforementioned methods. The agent capableof modulating cell growth or cell differentiation may intervene incellular transformation, or in immune dysregulation.

[0105] A further embodiment of the present invention is directed to amethod for identifying, characterizing or isolating an endogenousmolecule suspected of participating in the deadenylation or degradationof RNA or regulation thereof comprising

[0106] (A) providing the system of the present invention as describedabove;

[0107] (B) introducing said protein suspected of participating in theregulation of RNA turnover into said system;

[0108] (C) monitoring the stability of said target RNA sequence in saidsystem; and

[0109] (D) identifying, characterizing or isolating said endogenousmolecule able to modulate said deadenylation or degradation as capableof participating in the deadenylation or degradation of RNA orregulation thereof.

[0110] The molecule suspected of participating in the deadenylation ordegradation of RNA or regulation thereof may be protein or RNA.

[0111] In another embodiment of the present invention, a method isprovided for identifying an agent capable of modulating the degradationa target RNA sequence in the absence of deadenylation comprising

[0112] (A) providing a cell extract in the presence of a nucleotidetriphosphate;

[0113] (B) introducing said agent into said cell extract; and

[0114] (C) monitoring the degradation of said target RNA sequence insaid extract.

[0115] The present invention is also directed to kits for monitoring thestability of a preselected target RNA sequence under conditions capableof recapitulating regulated RNA turnover. Such kits comprise:

[0116] (a) cell extract optionally depleted of activity of proteins thatbind polyadenylate;

[0117] (b) other reagents; and

[0118] (c) directions for use of said kit.

[0119] A kit may further comprising nucleotide triphosphates, a reactionenhancer, a target RNA sequence, RNA binding proteins, RNA stabilitymodifiers, or any combination thereof. It will be seen by the skilledartisan that the kits of the invention provide the components forcarrying out the various methods disclosed herein, such as identifyingagents and endogenous factors that modulate RNA turnover, identifyingagents which modulate the RNA turnover activity of various factorsinvolved in RNA turnover, and others, in particular use in the screeningof small molecules for identifying potentially useful therapeutic agentsfor the prophylaxis and/or treatment of various conditions or diseasesbenefitted by modulating RNA turnover. The kits may be prepared toinvestigate either RNA deadenylation, RNA degradation, or both,depending on the components as described above. Furthermore, the cellextract may be partially purified. The kit may include reagents fordepleting activity of proteins present in the extract which bindpolyadenylate; such reagents, such as polyadenylate, polyadenylate boundto a matrix, an antibody to proteins that bind polyadenylate, and suchan antibody bound to a matrix.

[0120] The present invention may be better understood by reference tothe following non-limiting Examples, which are provided as exemplary ofthe invention. The following examples are presented in order to morefully illustrate the preferred embodiments of the invention. They shouldin no way be construed, however, as limiting the broad scope of theinvention.

EXAMPLE I ELAV Proteins Stabilize Deadenylated Intermediates in a NovelIn Vitro mRNA Deadenylation/Degradation System

[0121] Set forth herein is a novel in vitro mRNA stability system usingHela cell cytoplasmic S100 extracts and exogenous polyadenylated RNAsubstrates that reproduces regulated aspects of mRNA decay (turnover).The addition of cold poly(A) competitor RNA activated both asequence-specific deadenylase activity in the extracts as well as apotent, ATP-dependent ribonucleolytic activity. The rates of bothdeadenylation and degradation were up-regulated by the presence of avariety of AU-rich elements in the body of substrate RNAs. Competitionanalyses demonstrated that trans-acting factors were required for RNAde-stabilization by AU-rich elements. The ˜30 kDa ELAV protein, HuR,specifically bound to RNAs containing an AU-rich element derived fromthe TNF-α mRNA in the in vitro system. Interaction of HuR with AU-richelements, however, was not associated with RNA destabilization.Interestingly, recombinant ELAV proteins specifically stabilizeddeadenylated intermediates generated from the turnover of AU-richelement-containing substrate RNAs. Thus, mammalian ELAV proteins play arole in regulating mRNA stability by influencing the access ofdegradative enzymes to RNA substrates.

[0122] The relative stability of mRNA is an important regulator of geneexpression. The half-life of a specific mRNA can play a role indetermining both its steady state level of expression, as well as therate at which its gene product is induced (reviewed in Ross, 1995;Caponigro and Parker, 1996). Furthermore, mutations that affect thestability of mRNAs encoding regulatory factors can promote oncogenictransformation and immune dysregulation (Aghib et al., 1990; Schiavi etal., 1992). In general, many short-lived proteins, including thosederived from cytokines and proto-oncogenes, are encoded by short-livedmRNAs. Several mRNAs that encode stable proteins, such as a-globin, havealso been shown to have extraordinarily long half-lives (Holcik andLiebhaber, 1997). In addition, surveillance mechanisms that identify andreduce the half-lives of aberrant mRNAs that contain nonsense codonmutations have been described (Maquat, 1995; Jacobson and Peltz, 1996).Therefore, regulation of the half-life of mRNAs can have dramaticconsequences on cellular responses and functional outcomes during growthand development.

[0123] Through the application of genetics, the mechanisms and factorsinvolved in the turnover of mRNA in Saccharomyces cerevisiae arebeginning to be identified. Multiple pathways of mRNA turnover arepresent in yeast, allowing for numerous levels of regulation andfine-tuning of gene expression. One general pathway of mRNA decayinvolves poly(A) tail shortening followed by decapping and 5′-to-3′exonucleolytic decay (Muhlrad et al., 1994). A second general pathwayinvolves deadenylation followed by 3′-to-5′ turnover of the body of themRNA (Anderson and Parker, 1998). Endonucleolytic cleavage of some mRNAshas also been demonstrated (Presutti et al., 1995). Finally, anotheralternative decay pathway that bypasses deadenylation is involved in thetranslation-dependent degradation of nonsense codon-containing mRNAs(Weng et al., 1997). Several degradation enzymes and regulatory proteinsthat play a role in mRNA stability in yeast have been identified(Caponigro and Parker, 1996; Weng et al., 1997). Functionallysignificant interactions between the cap structure and the 3′ poly(A)tail of yeast mRNAs have also been described (Tarun and Sachs, 1997).Whether these observations are generally applicable to mammalian cells,however, remains to be established.

[0124] In vivo observations are beginning to allow some generalizationsconcerning major pathways of mRNA turnover in mammalian cells. A poly(A)tail of approximately 200 bases is added to most mRNAs during processingin the nucleus (Colgan and Manley, 1997). The poly(A) tail serves atleast two known functions in mRNA stability. First, in association withpoly(A) binding proteins (Bernstein et al., 1989; Ford et al., 1997), itprotects the mRNA from 3′-to-5′ exonucleases. Second, the poly(A) tailserves as an initiation site for the turnover of the mRNA. The poly(A)tail can be progressively shortened throughout the lifetime of a mRNA inthe cytoplasm. Controlling the rate of deadenylation appears to be animportant regulatory point in mRNA stability (Wilson and Treisman, 1988;Xu et al., 1997). Once the poly(A) tail is shortened to approximately30-65 bases, the body of the mRNA appears to be degraded in a rapidfashion in vivo without the accumulation of discernible intermediates(Chen et al., 1995; Xu et al., 1997). Little is known, however,concerning the enzymes and regulatory components involved in mammalianmRNA turnover.

[0125] In addition to the poly(A) tail, several cis-acting elements havebeen shown to play a role in mRNA stability. The 5′ terminal capstructure protects the transcript from exonucleases (Furuichi et al.,1977). Several destabilizing elements (Caput et al., 1986; Shyu et al.,1989; Bonnieu et al., 1990; Peng et al., 1996), as well as stabilizingelements (Stefonovic et al., 1997), located in the body of the mRNA havealso been identified. One well-characterized element that regulates mRNAstability is an AU-rich sequence (ARE) found in the 3′ untranslatedregion of many short-lived mRNAs (Shaw and Kamen, 1986). These AREsprimarily consist of AUUUA repeats or a related nonameric sequence(Lagnado et al., 1994; Zubiaga et al., 1995; Xu et al., 1997) and havebeen divided into three classes based on sequence characteristics anddegradation kinetics (Xu et al., 1997). In general, AREs have been shownto increase the rate of deadenylation and RNA turnover in atranslation-independent fashion (Chen et al., 1995; Fan et al., 1997).The underlying mechanism behind ARE function, however, remains to bedetermined.

[0126] Numerous proteins have been described that can bind in vitro toAU-rich elements (e.g. Malter, 1989; Vakalopoulou et al., 1991; Bohjanenet al., 1991; Brewer, 1991; Levine et al., 1993; Hamilton et al., 1993;Katz et al., 1994; Nakagawa et al., 1995; Ma et al., 1996), but theexact role of each factor in the process of mRNA turnover remains to bedefined. The ELAV family of ARE-binding proteins is evolutionarilyconserved and differentially expressed in tissues throughout thedevelopment of vertebrates (reviewed in Antic and Keene, 1997). AlthoughELAV proteins have been found in both the cytoplasm and the nucleus (Gaoand Keene, 1996), the most ubiquitously expressed form, HuR, can shuttlebetween the nucleus and the cytoplasm (Fan and Steitz, 1998; Peng etal., 1998; Atasoy et al, 1998). ELAV proteins play an important role ingrowth and development, as the Drosophila homolog is geneticallyessential for development and maintenance of the nervous system (Camposet al., 1985; Robinow and White, 1988). In addition, mammalian ELAVproteins are induced during differentiation and are distributed in RNPgranules along dendrites (Gao and Keene, 1996). Several lines ofevidence suggest that ELAV proteins control aspects ofpost-transcriptional gene expression (Gao and Keene, 1996; Koushika etal., 1996; Myer et al., 1997; Ma et al., 1997; Antic and Keene, 1998).Over-expression of ELAV family members, for example, has been shown toaffect accumulation of selected mRNAs (Jain et al., 1997; Levy et al.,1998; Fan and Steitz, 1998; Peng et al., 1998). The precise role of ELAVproteins and other ARE-binding factors, however, remains to beestablished.

[0127] Mechanistic questions in mammalian cells are usually bestapproached using biochemical systems due to the inherent difficultieswith mammalian cells as a genetic system. It has been difficult,however, to establish a versatile in vitro system to study mRNAstability and turnover. Based on in vivo observations and practicalconsiderations, an optimal in vitro system to study the process of mRNAstability should have the following properties: First, the system shouldbe efficient and highly reproducible. Second, minimal amounts(preferably undetectable) of RNA degradation in the system should be dueto random degradation by nonspecific contaminating ribonucleases. Third,deadenylation should occur before general degradation of the mRNA bodyis observed. Fourth, degradation of the mRNA body should occur in anapparently highly processive fashion without detectable intermediates.Fifth, regulation of the rate of overall deadenylation and degradationshould be observed in a sequence-specific manner. Finally, the systemshould work on exogenous RNAs to allow ease of experimentalmanipulation.

[0128] Reported herein is the discovery of a new and useful in vitromRNA stability system using cytoplasmic S100 extracts that fulfills allof the criteria listed above and possesses all of the properties knownto be involved in ARE-mediated mRNA turnover. This system has beensuccessfully used to demonstrate a role for the AU-rich element bindingproteins of the ELAV family in mRNA stability. These findings indicatethat ELAV proteins can affect a default pathway of ARE-mediateddegradation by either protecting the mRNA from nuclease attack or bydisplacing factors that otherwise mark these short-lived transcripts fordegradation. This in vitro system allows the identification of cellularfactors involved in mRNA turnover and help elucidate mechanisms involvedin the post-transcriptional regulation of gene expression.

[0129] Moreover, the in vitro system of the invention has readyapplications in high throughput assays to screen libraries of compoundsto elucidate which compounds may have applications as pharmaceuticalswhich can modulate the stability and turnover of RNA transcripts invivo, and thus be used to treat a wide variety of disease or disorders.

[0130] i. Development of an In vitro System that Deadenylates andDegrades RNA Substrates

[0131] The development of an in vitro system to study mRNA turnoverrequires the generation of a convenient source of poly(A)⁺ RNA substrateand an active cellular extract. In order to obtain substrate RNAs thatwere both polyadenylated and easy to identify using standard acrylamidegel technology, a novel and versatile ligation-PCR approach that canattach a template encoding a 60 base poly(A) tail to the 3′ end of DNAfragments that contain a Hind III site was used, and is described infra.In initial studies to develop an in vitro RNA stability system, a 60base poly(A) tail was attached to a 54 base polylinker-derived sequence(Gem-A60). The small size of this polyadenylated transcript made it easyto analyze intermediates in the pathway of RNA turnover on acrylamidegels. Cellular extracts were prepared following a standard cytoplasmicS100 protocol (Dignam et al., 1983) using hypotonically lysed Helaspinner cells with minor variations as described in the Materials andMethods.

[0132] Gem-A60 RNA was incubated in S100 extracts in the presence ofATP. As seen in FIG. 1A (left panel), very little turnover of theGem-A60 RNA was noted after 60 minutes of incubation. This reproducibleslow rate of turnover prompted us to hypothesize that an inhibitor ofthe deadenylation/degradation process might be present in S100 extracts.This hypothesis was based on several observations. First, previous workwith nuclear extracts determined that poly(A) binding proteins werestrong inhibitors of a 3′-to-5′ exonuclease activity (Ford et al.,1997). Second, the activity of a partially purified mammaliandeadenylase preparation was inhibited by high amounts of PABP (Kornerand Wahle, 1997). Third, over-expression of PABP in Xenopus oocytesinhibits maturation-specific deadenylation (Wormington et al., 1996). Inorder to test whether excess amounts of poly(A) binding proteins wereresponsible for inhibiting the deadenylation of Gem-A60 RNA in S100extracts, increasing amounts of cold poly(A) competitor RNA were addedto the reaction mixtures to sequester poly(A) binding proteins. As shownin FIG. 1A (right side), the addition of poly(A) competitor activated adegradation activity in the S100 extracts. The Gem-A60 RNA was shortenedto a species slightly larger than the size of a deadenylated marker(Gem-A0) and approximately 30% of the input RNA was degraded. Titrationexperiments performed in coordination with UV cross-linking studiesdemonstrated that the amount of poly(A) competitor RNA required toactivate the S100 extract precisely corresponded with the ability of thecompetitor to inhibit binding of proteins to the poly(A) tail of thesubstrate RNA (data not shown). Furthermore, the nucleolytic activitiesactivated by the addition of cold poly(A) RNA as competitor to the S100extracts were still observable at concentrations of poly(A) >500 ng.These data suggest that the activated nuclease(s) is highly refractoryto competition by poly(A).

[0133] The progressive shortening of the Gem-A60 RNA substrate observedupon incubation in S100 extract supplemented with poly(A) competitor RNAwas determined to be due to a 3′-to-5′, poly(A) tail-specificexonuclease based on the following observations: First, RNA substrates³²P-labeled exclusively at their 5′ cap structures were progressivelyshortened in the system in a similar fashion as uniformly labeledtranscripts (compare FIGS. 1A and 1B). These data suggest that theshortening of the input RNA occurred in a 3′-to-5′ direction. Thisconclusion was confirmed by separately analyzing the 5′ and 3′ portionsof RNA products from the in vitro system by RNAse H digestion prior togel electrophoresis. As shown in FIG. 1C, the 3′ portion of thesubstrate RNA (which consists primarily of the 60 base poly(A) tail) wasclearly being degraded before any turnover of the 5′ portion of thetranscript was detected. After 9 minutes of incubation, 72% of the 3′fragment containing the poly(A) tail is degraded, while only 19% of the5′ fragment has been turned over. Finally, in order to ascertain whetherthis 3′-to-5′ exonuclease activity was indeed a poly(A)-specificdeadenylase, we added 15 bases of non-adenylate sequence onto the 3′ endof the Gem-A60 RNA (Gem-A60-15). As seen in FIG. 1D, while the Gem-A60transcript (which contains a 3′ poly(A) tail) is an excellent substratefor the 3′ exonuclease activity, the Gem-A60-15 RNA, which has itspoly(A) tract internalized 15 bases, was not.

[0134] From these data it has been concluded that the addition ofpoly(A) competitor RNA to an S100 extract activates a deadenylase whichis active on exogenous, poly(A)+ substrate RNAs. The in vitro systemreproduces several aspects of mRNA stability observed in vivo. Thesurprising observation that the deadenylase itself is not apparentlyinhibited by cold poly(A) suggests that the native enzyme may not havehigh affinity for its substrate. The deadenylase activity may containadditional RNA binding activities that anchor it to mRNAs, perhaps aspart of a multi-component complex.

[0135] ii. RNA Turnover in the in vitro System is Regulated by AU-RichInstability Elements.

[0136] It was determined whether the RNA turnover activities exhibitedby the S100 extract system could be influenced or modulated by sequencesin the body of the transcript in a specific manner. The relativestability of small polyadenylated RNAs containing either a 54 basepolylinker sequence (Gem-A60), a 34 base AU-rich element (ARE) fromTNF-α mRNA (ARE-A60), or a 72 base ARE from the c-fos mRNA (Fos-A60) wasdetermined in the in vitro stability system. As shown in FIGS. 2A and2B, the turnover of both of the ARE-containing RNAs was dramaticallyincreased compared to the Gem-A60 control transcript. To directly assesswhether regulation by AREs was occurring in a sequence-specific fashion,the TNF-α-ARE was extensively mutated as described in Materials andMethods. Similar mutations in AU-rich instability elements were shownpreviously to greatly increase mRNA half-life in vivo (Myer et al.,1997). As seen in FIG. 2C, mutations in the ARE reduced the rate andextent of deadenylation /degradation over 3-fold in the in vitro system.Thus, RNA turnover in the in vitro system can be regulated or modulatedby AU-rich instability elements in a sequence-specific fashion.

[0137] All of the RNA substrates we have examined above contain a bodyof approximately 50-70 bases attached to a poly(A) tail. It was thendetermined whether regulated turnover using larger polyadenylated RNAsubstrates could be detected in the system of the invention. As shown inFIG. 2D, a polyadenylated 250 base RNA derived from the 3′ UTR of theSV40 late mRNA (SV-A60) was deadenylated but inefficiently degraded inthe in vitro system. Adding the TNF-α-ARE to the 3′ portion of this RNA(SVARE-A60) resulted in an approximate 3.5 fold increase in the rate ofturnover. Finally, a nearly full length (˜950 base) version of the humanGM-CSF mRNA was prepared, as well as one in which the ARE was deleted(GM−CSF(−ARE)). The 3′ ends of these transcripts were polyadenylatedusing yeast poly(A) polymerase (Martin and Keller, 1998). Gel purifiedRNAs were incubated in the in vitro stability system and aliquots wereremoved at the times indicated. As seen in FIG. 2E, the version of theGM-CSF mRNA that contains an ARE was approximately 2.5 fold less stablethan GM−CSF(−ARE) in the in vitro system. As seen above with othertranscripts, the GM-CSF transcripts were also deadenylated in thesystem. Deadenylation was not observable in FIG. 2E due to the lack ofresolution of the gel system employed, but can be observed usingformaldehyde-agarose gels (data not shown).

[0138] iii. Degradation but not Deadenylation Requires ATP

[0139] Transcripts with 60 adenylates at the 3′ end were observed toundergo both deadenylation and turnover in the in vitro system. This isconsistent with in vivo observations that suggest the poly(A) tail isshortened to about 30-65 bases before mRNA turnover is observed (Xu etal., 1997). Since degradation appeared to begin before the inputtranscript was completely deadenylated (eg. FIG. 2), it was difficult toquantitatively assess the effects of AU-rich elements on relativedeadenylation rates. In order to try uncoupling these processes andaccurately evaluate the effect of AREs on deadenylation rates in the invitro system, we surveyed the cofactor requirements that might be uniqueto either deadenylation or turnover. Both processes were inhibited bythe addition of EDTA (data not shown), suggesting a role for divalentcations. Curiously, deadenylation could occur without the addition ofATP/phosphocreatine to the system (FIG. 3A). Degradation, on the otherhand, required ATP/phosphocreatine as indicated by the accumulation ofdeadenylated intermediates in its absence (FIG. 3A, lanes -ATP). Byomitting ATP from the reaction, therefore, we were able to evaluaterelative deadenylation rates in the presence or absence of an AU-richinstability element. RNAs with physiological length poly(A) tails(150-200 bases) which lack (SV-A150-200) or contain (SVARE-A150-200) anARE were incubated in the in vitro system and aliquots were analyzed atthe times indicated. As seen in FIG. 3B, RNA substrates containing anARE were deadenylated at an approximately two fold faster rate than RNAsthat do not contain the instability element.

[0140] In summary, an in vitro mRNA stability system has been discoveredthat acts on exogenous substrates and faithfully reproduces all of theknown in vivo aspects of turnover. RNAs are first deadenylated prior todegradation of the body of the transcript. Degradation of the body ofthe mRNA then occurs in an apparently highly processive fashion with nodiscernible intermediates. Deadenylation and decay rates are increasedseveral fold by the inclusion of an AU-rich instability element. AREregulation of RNA stability is sequence-specific and highlyreproducible, as all three of the AREs we have tested in the in vitrosystem function in a similar fashion. This system should provide avaluable means to elucidate mechanistic aspects of regulated and generalmRNA turnover pathways.

[0141] iv. The Role of ARE-Binding Proteins in the in vitro System.

[0142] The in vitro system described here allows evaluation of the roleof ARE-binding proteins in the process of RNA deadenylation/degradation.Several proteins were found to be associated with ARE-containing RNAs inour extracts. As seen in FIG. 4A, a protein of ˜30 kDa and a group of˜40 kDa proteins were specifically UV cross-linked to the short ARE-A60transcript. A species of approximately 70 kDa was also detected whenthis ARE was inserted into a larger transcript (SVARE-A60; see FIG. 5B).It is possible that this 70 kDa protein was not detected on the ARE-A60RNA because of the relatively small size of the transcript. Efforts todetermine the identity of these cross linked species using availableantibodies to known ARE-binding proteins revealed the presence of anELAV protein. As shown in FIG. 4B, immunoprecipitation assays identifiedthe 30 kDa protein as HuR (a.k.a. HuA), a member of the ELAV proteinfamily that is ubiquitously expressed in all tissues (Good, 1995; Ma etal., 1996; Myer et al., 1997). Antisera against another RNA-bindingprotein of approximately 30 kDa, hnRNP A1, failed to detect any crosslinked protein in our system (FIG. 4B). Two additional antisera weretested in order to identify the 40 kDa band. Antibodies to hnRNP Cprotein failed to detect any cross linked protein, while antisera toAUF-1 (a.k.a. hnRNP D)(Brewer, 1991) did precipitate a small amount ofcross linked 40 kDa protein (data not shown). However, this cross linkedproduct was not competed by increasing amounts of a 34 base syntheticARE competitor RNA (data not shown). The significance of this low levelof non-specific AUF-1 cross linking in the system is unclear. It wasconcluded that the 30 kDa species that specifically cross links to theARE element is HuR, a protein that has been previously suggested to playa role in ARE-mediated mRNA decay (Vakaloloupou et al, 1991; Antic andKeene, 1997; Myer et al., 1997).

[0143] Next, it was determined whether the interaction of the crosslinked ARE binding proteins with the element was required to mediateinstability. Synthetic ribonucleotides containing either a 34 base TNF-αARE or randomly chosen, non-ARE sequences were used. Syntheticcompetitor RNAs were added in increasing amounts to the in vitrostability system and their effect on RNA turnover was assessed. As seenin FIG. 5A, the ARE competitor RNA completely inhibited deadenylationand degradation at 40 pm, while the non-specific RNA had no effect atsimilar concentrations. The ARE competitor RNA had a similar effect onthe deadenylation/degradation of RNAs whether or not they contained anARE. Thus, factors capable of interacting with AREs are important fordeadenylation, and may be a part of a multi-proteindeadenylase/degradation complex.

[0144] The ability of the synthetic ARE competitor RNA to blockdeadenylation was compared with the ability of the RNA to compete forinteraction of ARE-binding proteins with the substrate transcript. EDTAwas added to cross-linking assays to inhibit RNA turnover and toevaluate the effect of various levels of competitor oncross-linking/label transfer efficiency. As shown in FIG. 5B, allARE-binding proteins (including HuR protein that could beimmunoprecipitated using specific antisera prior to gel electrophoresisas shown in panel C) were specifically competed from the SV-ARE-A60 RNAsubstrates upon addition of 5 pm of the synthetic RNA competitor. Asshown in FIG. 5A, however, 5 pm of synthetic ARE competitor RNA failedto have an appreciable effect on the rate of RNAdeadenylation/degradation in the system. Hence, none of the ARE-bindingproteins that could be detected by cross-linking appear to be requiredfor deadenylation/degradation in the in vitro system.

[0145] v. ELAV Proteins Prevent Degradation of Deadenylated Transcriptsin the in vitro System

[0146] Since the ARE binding proteins we detected by cross-linking donot appear to be required for deadenylation/degradation, they may play arole in transcript stability. Consistent with this model, recent in vivodata suggest that overexpression of He1-N1 and HuR proteins canstabilize ARE-containing transcripts (Jain et al., 1997; Fan and Steitz,1998; Peng et al., 1998). A mouse recombinant HuR protein, as well asother members of the ELAV family (He1-N1 and He1-N2 [a.k.a. HuB]) wereproduced as GST fusion proteins and added these to the in vitrostability system at a 10:1 molar ratio to substrate RNA. Similar datawere obtained using any of the three recombinant ELAV family proteins,and only data with rHe1-N

[0147]1 is shown. As seen in FIG. 6A, rHe1-N1 protein failed to affectdeadenylation of the SVARE-A60 RNA substrate in the in vitro system, butstabilized a deadenylated intermediate. GST alone, or another GST-fusionprotein that binds RNA (hnRNP H′) had no effect on transcript stabilityin the in vitro system (FIG. 6B). As a result, it was concluded that theELAV family of RNA binding proteins function to protect deadenylatedtranscripts from the degradation enzymes.

[0148] Next, it was tested whether the RNA substrate must contain an AREin order for rELAV proteins to stabilize a deadenylated intermediate inthe in vitro system. ARE-A60 RNA, or an unrelated but similarly sizedand polyadenylated transcript, CX-A60, were incubated in the in vitrosystem in the presence or absence of rELAV proteins. As seen in FIG. 6C,rHe1N1 (or other rELAV proteins [data not shown]) stabilized thedeadenylated intermediate only from RNAs that contain an ARE bindingsite. Thus, the stabilization of deadenylated intermediates by ELAVproteins requires an ARE. Furthermore, ELAV proteins can stabilize adeadenylated intermediate whether the ARE is located at the 3′, 5′ orcentral positions of the 250 base SVARE-A60 RNA. These data indicatethat the ARE-ELAV protein complex probably is not simply preventingturnover through steric blocking of an end of the transcript, therebypreventing exonuclease access.

[0149] Set forth herein is a novel and useful in vitro RNA stabilitysystem that faithfully reproduces many known aspects of in vivo mRNAturnover in mammalian cells. Exogenous RNA substrates are deadenylatedbefore degradation of the RNA body occurs in an apparently highlyprocessive fashion without detectable intermediates. Furthermore, therates of RNA deadenylation and degradation are regulated by AU-richelements in the system in a sequence-specific manner. The system of theinvention has been successfully used to determine a role for the ELAVfamily of ARE binding proteins in the stability of deadenylatedtranscripts by specifically blocking the degradation step. These dataillustrate the value of the system to address the mechanism of regulatedmRNA turnover.

[0150] The in vitro system described in this report has several keytechnical advantages that significantly increase its utility. First, thesystem is highly reproducible and uses standard S100 cytoplasmicextracts from Hela spinner cells. In fact, nine independent preparationsof S100 extract that all function in the assay in a similar fashion havebeen tested. The only difference among extracts appears to be in thekinetics of turnover (e.g. compare the slight differences in the patternof turnover of Gem-A60 RNA in FIG. 1A with the pattern observed in FIG.1D). Second, the extracts exhibit minimal background degradation of RNAdue to non-specific nucleases. This lack of noise in the systemsignificantly contributes to its reproducibility. Another key element ofthe system is that is uses exogenous polyadenylated RNAs as substrates.This property affords variety in RNA substrate preparation and sequencemanipulation. Fourth, the system exhibits sequence-specific regulationby AU-rich elements in the absence of translation. In total, thesetechnical advantages make the system a valuable reagent to identifycomponents involved in mRNA turnover and address the mechanism ofregulated mRNA stability.

[0151] The addition of poly(A) competitor RNA was required to activateS100 extracts to efficiently deadenylate and degrade RNAs in a regulatedmanner. Titration of cold poly(A) demonstrated that the extracts becameactivated for deadenylation/degradation when sufficient competitor wasadded to substantially reduce cross linking of a 70 kDa poly(A) bindingprotein to the poly(A) tail of the radiolabeled substrate RNA (data notshown).

[0152] Surprisingly, the deadenylation in the extracts remain activeeven in the presence of >500 ng of poly(A). Commercial poly(A)preparations prepared with polynucleotide phosphorylase, therefore, donot appear to be able to interact with and sequester the deadenylaseenzyme. These data suggest that the deadenylase activity is either inextraordinary concentrations in the extracts or may not have a strongaffinity for its substrate. In conjunction with this, it has beenobserved that an increase in deadenylation rate of ARE containing RNAs(FIGS. 2 and 3), as well as the ability of the ARE competitor RNA toinhibit deadenylation of non-ARE containing substrates. These datasuggest that ARE-binding proteins may be associated with the deadenylaseactivity.

[0153] Moreover, HuR protein, a ubiquitously expressed member of theELAV family of RNA binding proteins (Good, 1995; Ma et al., 1996; Myeret al., 1997; Antic and Keene, 1997), has been identified as one of themajor ARE binding factors in the system of the invention. Also, thesystem of the invention has been successfully used to detect weakbinding to AUF-1 (hnRNP D), a protein previously speculated to beinvolved in regulated mRNA decay in vitro (DeMaria and Brewer, 1996).AUF-1, therefore, does not appear to play a significant role intranscript instability in our system. ELAV proteins are not required fordeadenylation/degradation, but rather play a role in the stability ofdeadenylated RNAs that contain an ARE (FIG. 6). These data suggest thatin addition to its effect on deadenylation rates (Chen et al., 1995; Xuet al., 1997), the ARE influences the efficiency of turnover of the bodyof the mRNA. In vivo observations (Chen et al., 1995; Xu et al., 1997;Peng et al., 1998) also support the conclusion that ARE influences mRNAdegradation rates.

[0154] ELAV proteins, therefore, appear to regulate mRNA stability invitro, an observation consistent with in vivo transfection studies. TheELAV family comprises four members, three of which are expressed in atissue or developmental specific manner (reviewed in Antic and Keene,1997). Tissue-specific ELAV proteins are also localized primarily to thecytoplasm, while the ubiquitous HuR protein is predominantly nuclear andcan redistribute to the cytoplasm (Atasoy et al., 1998; Peng et al.,1998; Fan and Steitz, 1998). It has been suggested that differentiallyexpressed ELAV proteins play a role in regulating the stability of bothnuclear and cytoplasmic RNA, thereby fine tuning gene expression inspecific developmental states (Gao and Keene, 1996; Antic and Keene,1998).

[0155] The competition data shown in FIG. 5 clearly demonstrate thatfactors associated with the ARE are required fordeadenylation/degradation of substrate RNAs. Based on the kinetics ofcompetition, these factors must either be much more abundant than thecross-linkable ARE binding proteins like HuR, or interact with the AREwith a much lower affinity. We favor the latter model, and suggest thatthese factors are part of a multi-component complex that includes thedeadenylase and degradation enzymes. Through multiple cooperativeinteractions, these weak ARE binding components may allow efficientassembly of the deadenylase/degradation complex on ARE containingtranscripts while still allowing the complex to assemble, albeit lesseffectively, on non-ARE containing RNAs. The RNA binding components ofthis proposed complex also may have affinity for other non-AREinstability elements (e.g. Peng et al., 1996).

[0156] The observation that endogenous HuR protein in S100 extracts setforth herein can be cross-linked to ARE-containing RNA substrates (FIG.5) makes it surprising that an ARE can function as a destablizingelement in the in vitro assay. Since HuR protein is predominantlynuclear, however, only low levels of the protein are likely to bepresent in our cytoplasmic extracts. This low level of HuR protein isprobably unable to efficiently compete with destablizing factors forbinding to the ARE. In fact, sequestration of the HuR protein by theaddition of low levels of synthetic ARE competitor RNA does lead to anincreased rate of turnover of ARE-containing RNAs in the in vitrosystem. As shown in FIG. 5A, the amount of SVARE-A60 RNA remaining after30 min. in the system in the absence of competitor RNA (lane 0) isapproximately 20% greater than when the assay is done in the presence of5 pm of ARE competitor RNA (lane 5 pm). The removal or sequestration ofHuR protein in S100 extracts, therefore, may be necessary in order toobserve regulated deadenylation and degradation in some instances.

[0157] Materials and Methods

[0158] Transcription Templates and RNAs

[0159] RNAs were produced by in vitro transcription using SP6 polymerase(Melton et al., 1984) in the presence of ^(7m)GpppG cap analog andradiolabeled UTP or ATP as indicated. All transcripts were gel purifiedprior to use. For RNAs labeled exclusively at the 5′ cap, transcriptionreactions were performed in the absence of cap analog and radioactivenucleotides. Capping was then performed using guanyltransferase (BRL)and radiolabled GTP according to the manufacturer's recommendations. Thesequence of short RNAs used as substrates in the in vitro system isshown in Table 1.

[0160] Transcription templates were derived as follows (Please note thatall synthetic oligonucleotides used as transcription templates shownbelow contain a 24 base SP6 promoter fragment at their 5′ ends): Gem-A0RNA was produced from Hind III cut pGem4 (Promega). Gem-A60-15 RNA wasproduced from the PCR product used to produce Gem-A60 RNA (see below)without removing the primer binding site with Ssp I. Templates forARE-A0 RNA were generated by hybridizing the synthetic oligonucleotide 5′-ATTTAGGTGACACTATAGAATACACATTATTTATTATTTATTTATTATTTATTTA TTTA-3′ (SEQID NO: 1) and its appropriate complement. Templates for MT-ARE-A0 RNAwere generated by hybridizing the synthetic oligonucleotide5′-ATTTAGGTGACACTATAGAATACACGTTAGTATTCATTTGTTTACTATTGATTTC TTTA-3′ (SEQID NO:2) and its appropriate complement. Templates for Fos-A0 RNA weregenerated by hybridizing the synthetic oligonucleotide5′-ATTTAGGTGACACTATAGAATACACAAATTTTATTGTGTTTTTAATTTATTTATTAAGATGGATTCTC-3′ (SEQ ID NO:3) and its appropriate complement. Thetemplate for SV-A0 RNA was Hind III cut pSVL-Gem (Wilusz et al., 1988).Templates for SVARE-A0 RNA were generated by inserting the TNF-α AREcontaining oligonucleotide 5′-ATTATTTATTATTTATTTATTATTTATTATTTA (SEQ IDNO:4) and its appropriate complement between the PstI and Hind III sitesof pSVL-Gem (located near the 3′ end of the RNA). SVARE-A0 RNA wastranscribed from Hind III linearized DNA. The template for GM−CSF(+ARE)RNA was EcoRI cut pGM-CSF (Shaw and Kamen, 1986). The template forGM−CSF(−ARE) RNA was NcoI cut pGM-CSF. Templates for CX-A0 RNA weregenerated by hybridizing the synthetic oligonucleotide 5′-ATTTAGGTGACACTATAGAATACACCCCAACGGGCCCTCCCTCCCCTCCTTGCACCATCATCGCATCACG (SEQ ID NO:5) and its appropriate complement.

[0161] Synthetic RNAs used in competition studies were made by the NJMSMolecular Core Facility and contained the following sequences: ARE:

[0162] 5′AUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUUA (SEQ ID NO:6);

[0163] Non-specific competitor: 5′-GUCACGUGUCACC (SEQ ID NO:7).

[0164] Addition of Poly(A) Tails to Transcripts

[0165] A template for a 60 base poly(A) tail was added to DNA templatesusing a ligation/PCR protocol have recently been described (Ford et al.,1997). Briefly, all of the templates described above contain a Hind IIIsite that is used to generate the 3′ end of the RNA. The syntheticoligonucleotide 5′-AGCTA₆₀TATTGAGGTGCTCGAGGT (SEQ ID NO:8) and itsappropriate complement were generated, hybridized, and ligated to HindIII cut DNA templates. Ligation products were amplified using an SP6promoter primer (5′-CATACGATTTAGGTGACACTATAG (SEQ ID NO:9)) and a primerspecific for the 3′ end of the ligated oligonucleotide(5′-ACCTCGAGCACCTC (SEQ ID NO:10)). Amplified products were purified onCentricon 100 columns, cut with SspI, and used as templates for SP6polymerase generate RNAs carrying the ‘A60’ designation. Poly(A)polymerase (Amersham) was used to add 150-200 base poly(A) tails ontotranscripts. RNAs were incubated with enzyme according to themanufacturer's recommendations on ice for 5-8 min. Following thereaction, RNAs were extracted with phenol-chloroform, ethanolprecipitated, and purified on 5% acrylamide gels containing 7M urea toobtain RNAs with the appropriate amount of poly(A) at the 3′ end.

[0166] S100 Extract Production

[0167] Cytoplasmic extracts were prepared from Hela spinner cells grownin JMEM supplemented with 10% horse serum as described by Dignam et al(1983) with the following two modifications. First, followingcentrifugation at 100,000×g for 1 hr, the supernatant was adjusted to10% glycerol prior to dialysis. Second, dialysis times were shortened to30 min. Extracts were stored at −80° C.

[0168] In vitro RNA Deadenylation/Degradation System

[0169] Typically, approximately 200,000 cpm (˜50 fm) of gel purified RNAis used per reaction. In comparative studies, equal molar amounts oftranscripts were used. A typical 14.25 μl reaction mixture contains 3.25μl of 10% polyvinyl alcohol, 1 μl of a 12.5 mM ATP/250 mMphosphocreatine mixture, 1 μl of 500 ng/ul poly(A) (Pharmacia), 1 μl ofRNA and 8 μl of dialyzed extract. Reactions were incubated at 30° C. forthe times indicated and stopped by the addition of 400 μl of stop buffer(400 mM NaCl, 25 mM Tris-Cl, pH 7.6, 0.1% SDS). Reaction mixtures werephenol extracted, ethanol precipitated and analyzed on a 5% acrylamidegel containing 7M urea. All quantitation was performed using a MolecularDynamics Phosphorimager.

[0170] Recombinant ELAV proteins (HuR, He1-N1 and He1-N2) were made asGST-fusion proteins in E. coli and purified using glutathione-sepharoseaffinity chromatography according to the manufacturer's recommendations(Levine et al, 1993).

[0171] RNase H Digestion ARE-A60 RNA, radiolabeled at A residues, wasincubated in the in vitro stability system for the times indicated. RNAproducts were phenol extracted and concentrated by ethanolprecipitation. The sample was resuspended in a final volume of 30 μlcontaining 20 mM Tris-Cl, pH 8.0, 100 mM NaCl, 10 mM MgCl₂, 1 mM DTT,100 picomoles of the antisense oligonucleotide 5′-AGTTAAATAAAT (SEQ IDNO:11), and 1 unit of RNase H. Reactions were incubated at 37° C. for 30min. and products were analyzed on a 5% acrylamide gel containing 7Murea.

[0172] UV Cross Linking and Immunoprecipitations

[0173] UV cross linking/label transfer experiments were performed asdescribed previously using a Sylvania G15T8 germicidal light (Wilusz andShenk, 1988). Cross linking experiments were done in the presence of 25mM EDTA to inhibit RNA turnover to allow for accurate comparisonsbetween samples. Following digestion with RNAses A, T1 and T2, crosslinked proteins were analyzed on 10% acrylamide gels containing SDS.

[0174] For immunoprecipitation analysis following UV cross linking andRNAse treatment, 300 μl of RIPA buffer (0.15M NaCl, 1% NP-40, 0.5%deoxycholate, 0.1% SDS and 50 mM Tris-Cl, pH 7.6) was added to samples.Following a brief centrifugation in a microfuge, precleared samples wereincubated on ice with antibodies for 1 hr. Antigen-antibody complexeswere collected using formalin fixed, washed protein-A positive S. aureuscells, washed five times using RIPA buffer, and analyzed on a 10%acrylamide gel containing SDS. Antibodies specific for GRSF (Qian andWilusz, 1994) and hnRNP A1 (Wilusz and Shenk, 1990) have been describedpreviously. The preparation and characterization of rabbit polyclonalantibodies specific for HuR will be described elsewhere (Atasoy et al.,1998).

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[0236] The present invention is not to be limited in scope by thespecific embodiments describe herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

[0237] It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

[0238] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties. SEQUENCE IDS:601-1-088 5′-ATTTAGGTGACACTATAGAATACACATTATTTA (SEQ ID NO:1)TTATTTATTTATTATTTATTTATTTA-3′ 5′-ATTTAGGTGACACTATAGAATACACGTTAGTAT (SEQID NO:2) TCATTTGTTTACTATTGATTTCTTTA-3′5′-ATTTAGGTGACACTATAGAATACACAAATTTTA (SEQ ID NO:3)TTGTGTTTTTAATTTATTTATTAAGATGGATTCTC- 3′5′-ATTATTTATTATTTATTTATTATTTATTATTTA (SEQ ID NO:4)5′-ATTTAGGTGACACTATAGAATACACCCCAACGG (SEQ ID NO:5)GCCCTCCCTCCCCTCCTTGCACCATCATCGCATCAC G5′`AUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUU (SEQ ID NO:6) A 5′-GUCACGUGUCACC.(SEQ ID NO:7) 5′-AGCTA₆₀TATTGAGGTGCTCGAGGT (SEQ ID NO:8)5′-CATACGATTTAGGTGACACTATAG (SEQ ID NO:9) 5′-ACCTCGAGCACCTC (SEQ IDNO:10) 5′-AGTTAAATAAAT (SEQ ID NO:11) AUUUA (SEQ ID NO:12)

[0239]

1 12 1 59 DNA Artificial Sequence Description of Artificial Sequence Byhybridizing this synthetic oligonucleotide and its appropriatecomplement, template for ARE-A0 RNA were generated. 1 atttaggtgacactatagaa tacacattat ttattattta tttattattt atttattta 59 2 59 DNAArtificial Sequence Description of Artificial Sequence By hybridizingthis synthetic oligonucleotide and its appropriate complement ,templates for MT-ARE-A0 RNA were generated. 2 atttaggtga cactatagaatacacgttag tattcatttg tttactattg atttcttta 59 3 68 DNA ArtificialSequence Description of Artificial Sequence By hybridizing thissynthetic oligonucleotide and its appropriate complement , templates forFos-A0 RNA were generated. 3 atttaggtga cactatagaa tacacaaatt ttattgtgtttttaatttat ttattaagat 60 ggattctc 68 4 33 DNA Artificial SequenceDescription for artificial sequence Templates for SVARE-A0 RNA weregenerated by inserting the TNF-alpha ARE containing this oligonucleotideand its appropriate complement between the PstI and Hind 4 attatttattatttatttat tatttattat tta 33 5 70 DNA Artificial Sequence Description ofArtificial Sequence By hybridizing this synthetic oligonucleotide andits appropriate complement , templates for CX-A0 RNA were generated. 5atttaggtga cactatagaa tacaccccaa cgggccctcc ctcccctcct tgcaccatca 60tcgcatcacg 70 6 34 RNA Artificial Sequence Description of ArtificialSequence Synthetic RNAs used in competition studies. ARE. 6 auuauuuauuauuuauuuau uauuuauuua uuua 34 7 13 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic RNA used in competition studiescontains this sequence. Non-specific competitior. 7 gucacguguc acc 13 823 DNA Artificial Sequence Description of Artificial Sequence Thissynthetic oligonucleotide and its appropriate complement were generated,hybridized, and ligatedto Hind III cut DNA templates. 8 agctatattgaggtgctcga ggt 23 9 24 DNA Artificial Sequence Description of ArtificialSequence SP6 promoter primer. 9 catacgattt aggtgacact atag 24 10 14 DNAArtificial Sequence Description of Artificial Sequence A specific 3′ endprimer for ligated oligonucleotide. 10 acctcgagca cctc 14 11 12 DNAArtificial Sequence Description of Artificial Sequence Antisenseoligonucleotide. 11 agttaaataa at 12 12 5 RNA Artificial SequenceDescription of Artificial Sequence This sequence often repeats in AREs(A-U rich sequence) found in the 3′ untranslated region of manyshort-lived mRNAs. 12 auuua 5

What is claimed is:
 1. An in vitro system capable of recapitulatingregulated RNA turnover of an exogenously added preselected target RNAsequence comprising a cell extract and said target RNA sequence.
 2. Thesystem of claim 1 wherein said regulated RNA turnover is selected fromthe group consisting of AU-rich element regulated RNA turnover andC-rich element regulated turnover.
 3. The system of claim 1 wherein saidcell extract is isolated from lysed eukaryotic cells or tissues.
 4. Thesystem of claim 3 wherein said cell extract is obtained from a cell lineselected from the group consisting of HeLa cells and a T cell line. 5.The system of claim 1 wherein said cell extract is prepared from cellscomprising foreign nucleic acid.
 6. The system of claim 1 wherein saidcell extract is prepared from cells which are infected, stablytransfected, or transiently transfected.
 7. The system of claim 1wherein said cell extract is partially purified.
 8. The system of claim1 wherein said cell extract is depleted of activity of proteins thatbind polyadenylate.
 9. The system of claim 8 wherein said cell extractdepleted of activity of proteins that bind polyadenylate is prepared bya method selected from the group consisting of: (a) addition to saidsystem of polyadenylate competitor RNA; (b) sequestration of proteinsthat bind polyadenylate; (c) addition of a proteinase that inactivates aprotein that bind to polyadenylate; and (d) addition of an agent thatprevents the interaction between polyadenylate and an endogenousmacromolecule that binds to polyadenylate
 10. The system of claim 9wherein said sequestration of proteins that bind polyadenylate isachieved by treatment of said extract with an material that depletesmacromolecules that bind polyadenylate selected from the groupconsisting of antibodies to proteins that bind polyadenylate,polyadenylate, and the combination thereof.
 11. The system of claim 10wherein said material is attached to a matrix.
 12. The system of claim 1wherein said target RNA sequence is selected from the group of syntheticRNA, naturally occurring RNA, messenger RNA, chemically modified RNA,and RNA-DNA derivatives.
 13. The system of claim 12 wherein said targetRNA sequence comprises a 5′ cap and a 3′ polyadenylate sequence.
 14. Thesystem of claim 1 wherein said target RNA sequence is selected from thegroup consisting of unlabeled target RNA sequence, labeled target RNAsequence, and the combination thereof.
 15. The system of claim 14wherein said labeled target RNA sequence is labeled with a moiety isselected from the group consisting of a fluorescent moiety, a visiblemoiety, a radioactive moiety, a ligand, and a combination of fluorescentand quenching moieties.
 16. The system of claim 1 additionallycomprising exogenously added nucleotide triphosphate.
 17. The system ofclaim 16 wherein said nucleotide triphosphate is ATP.
 18. The system ofclaim 1 further comprising a reaction enhancer.
 19. The system of claim18 wherein said reaction enhancer is selected from the group consistingof polyvinyl alcohol, polyvinylpyrrolidone and dextran.
 20. The systemof claim 19 wherein said reaction enhancer is polyvinyl alcohol.
 21. Amethod for identifying an agent capable of modulating the stability of atarget RNA sequence comprising (A) providine the system of claim 1; (B)introducing said agent into said system; (C) determining the extent ofturnover of said target RNA sequence; and (D) identifying an agent ableto modulate the extent of said turnover as capable of modulating thestability of said target RNA sequence.
 22. The method of claim 21wherein said system additionally comprises nucleotide triphosphate. 23.The method of claim 22 wherein said nucleotide triphosphate is ATP. 24.The method of claim 21 wherein said agent is an RNA stability modifyingmolecule.
 25. The method of claim 21 wherein said target RNA sequence isselected from the group consisting of unlabeled target RNA sequence,labeled target RNA sequence, and the combination thereof.
 26. The methodof claim 25 wherein said labeled RNA sequence is labeled with a moietyis selected from the group consisting of a fluorescent moiety, a visiblemoiety, a radioactive moiety, a ligand, and a combination of fluorescentand quenching moieties.
 27. The method of claim 21 wherein saidmonitoring the extent of turnover of said target RNA sequence comprisesdetermining the extent of degradation of said labeled target RNA. 28.The method of claim 21 wherein said modulating the stability of a targetRNA sequence increases the stability of said target RNA sequence. 29.The method of claim 21 wherein said modulating the stability of a targetRNA sequence decreases the stability of said RNA sequence.
 30. Themethod of claim 21 wherein said agent is capable of modulating theactivity of a AU rich element binding protein or a C-rich elementbinding protein.
 31. The method of claim 30 wherein said AU rich elementbinding protein is selected from the group consisting of a member of theELAV protein family; AUF1; tristetrapolin; AUH; TIA; TIAR;glyceraldehyde-3-phosphate; hnRNP C; hnRNP A1; AU-A; and AU-B.
 32. Themethod of claim 31 wherein said member of the ELAV protein family isselected from the group consisting of HuR, He1-N1, HuC and HuD.
 33. Amethod for identifying an agent capable of modulating the stability of atarget RNA sequence in the presence of an exogenously added RNAstability modifier comprising (a) providing the system of claim 1; (b)introducing said RNA stability modifier into said system; (c)introducing said agent into said system; (d) determining the extent ofturnover of said target RNA sequence; and (e) identifying an agent ableto modulate the extent of said turnover as capable of modulating thestability of said target RNA sequence in the presence of saidexogenously added RNA stability modifier.
 34. The method of claim 33wherein said system additionally comprises nucleotide triphosphate. 35.The method of claim 34 wherein said nucleotide triphosphate is ATP. 36.The method of claim 33 wherein said target RNA sequence is selected fromthe group consisting of unlabeled target RNA sequence, labeled targetRNA sequence, and the combination thereof.
 37. The method of claim 36wherein said labeled RNA sequence is labeled with a moiety is selectedfrom the group consisting of a fluorescent moiety, a visible moiety, aradioactive moiety, a ligand, and a combination of fluorescent andquenching moieties.
 38. The method of claim 33 wherein said determiningthe extent of turnover of said target RNA sequence comprises determiningthe extent of degradation of said labeled target RNA.
 39. The method ofclaim 33 wherein said RNA stability modifier increases the stability ofsaid target RNA sequence.
 40. The method of claim 39 wherein said agentdecreases the stability of said target RNA sequence increased by saidRNA stability modifier.
 41. The method of claim 33 wherein said RNAstability modifier decreases the stability of said target RNA sequence.42. The method of claim 41 wherein said agent increases the stability ofsaid target RNA sequence decreased by said RNA stability modifier. 43.The method of claim 33 wherein said agent is capable of modulating theactivity of a AU rich element binding protein or a C-rich elementbinding protein.
 44. The method of claim 43 wherein said AU rich elementbinding protein is selected from the group consisting of a member of theELAV protein family; AUF1; tristetrapolin; AUH; TIA; TIAR;glyceraldehyde-3-phosphate; hnRNP C; hnRNP A1; AU-A; and AU-B.
 45. Themethod of claim 44 wherein said member of the ELAV protein family isselected from the group consisting of HuR, He1-N1, HuC and HuD.
 46. Amethod for identifying an agent capable of modulating the deadenylationof a target RNA sequence comprising (A) providing the system of claim 1in the absence of a nucleotide triphosphate; (B) introducing said agentinto said system; (C) monitoring the deadenylation of said target RNAsequence in said system; and (D) identifying an agent able to modulatethe extent of said deadenylation as capable of modulating thedeadenylation of said target RNA sequence.
 47. A method for identifyingan agent capable of modulating the deadenylation and degradation of atarget RNA sequence comprising (A) providing the system of claim 1 inthe presence of a nucleotide triphosphate; (B) introducing said agentinto said system; (C) monitoring the deadenylation and degradation ofsaid target RNA sequence in said system; and (D) identifying an agentable to modulate the extent of said deadenylation and degradation ascapable of modulating the deadenylation and degradation of said targetRNA sequence.
 48. A method for identifying an agent capable ofmodulating cell growth or cell differentiation in a mammal comprisingdetermining the ability of said agent to modulate the stability of atarget RNA sequence involved in the modulation of cell growth ordifferentiation in accordance with claim
 19. 49. The method of claim 48wherein said agent capable of modulating cell growth or celldifferentiation intervenes in cellular transformation.
 50. The method ofclaim 48 wherein said agent capable of modulating cell growth or celldifferentiation intervenes in immune dysregulation.
 51. A method foridentifying, characterizing or isolating an endogenous moleculesuspected of participating in the deadenylation or degradation of RNA orregulation thereof comprising (A) providing the system of claim 1; (B)introducing said protein suspected of participating in the regulation ofRNA turnover into said system; (C) monitoring the stability of saidtarget RNA sequence in said system; and (D) identifying, characterizingor isolating said endogenous molecule able to modulate saiddeadenylation or degradation as capable of participating in thedeadenylation or degradation of RNA or regulation thereof.
 52. Themethod of claim 51 wherein said molecule suspected of participating inthe deadenylation or degradation of RNA or regulation thereof is proteinor RNA.
 53. A kit for monitoring the stability of a preselected targetRNA sequence under conditions capable of recapitulating regulated RNAturnover, said kit comprising: (a) cell extract depleted of activity ofproteins that bind polyadenylate; (b) other reagents; and (c) directionsfor use of said kit.
 54. The kit of claim 53 further comprisingnucleotide triphosphates, a reaction enhancer, a target RNA sequence, orany combination thereof.
 55. A method for identifying an agent capableof modulating the degradation a target RNA sequence in the absence ofdeadenylation comprising (A) providing a cell extract in the presence ofa nucleotide triphosphate; (B) introducing said agent into said cellextract; and (C) monitoring the degradation of said target RNA sequencein said extract.